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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210076	2/6	Rattus norvegicus	Urine	UF qEV SEC (non-commercial)	Hinzman CP	2022	63%
Study summary Full title An optimized method for the isolation of urinary extracellular vesicles for molecular phenotyping: detection of biomarkers for radiation exposure. All authors Hinzman CP, Jayatilake M, Bansal S, Fish BL, Li Y, Zhang Y, Bansal S, Girgis M, Iliuk A, Xu X, Fernandez JA, Griffin JH, Ballew EA, Unger K, Boerma M, Medhora M, Cheema AK Journal J Transl Med Abstract Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications ac (show more...)Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications across clinical research, including monitoring radiation exposure. A key limitation to their implementation is minimal standardization in EV isolation and analytical methods. Further, most urinary EV isolation protocols necessitate large volumes of sample. This study aimed to compare and optimize isolation and analytical methods for EVs from small volumes of urine. (hide) EV-METRIC 63% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Ultrafiltration Commercial method Size-exclusion chromatography (non-commercial) Protein markers EV: TSG101/ ANXA5/ CD81/ Alix/ Flotillin1/ EpCAM/ CD63 non-EV: GM130 Proteomics no Show all info Study aim New methodological development/Biomarker/Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Urine Separation Method Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Commercial kit qEV Size-exclusion chromatography Total column volume (mL) 3.5 Sample volume/column (mL) 0.15 Resin type Not Specified Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ CD63/ EpCAM/ ANXA5/ TSG101/ Alix/ CD81 Detected contaminants GM130 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 156.5 EV concentration Yes EM EM-type Cryo-EM Image type Close-up

	EV210076	3/6	Rattus norvegicus	Urine	Other/ EVTrap	Hinzman CP	2022	63%
Study summary Full title An optimized method for the isolation of urinary extracellular vesicles for molecular phenotyping: detection of biomarkers for radiation exposure. All authors Hinzman CP, Jayatilake M, Bansal S, Fish BL, Li Y, Zhang Y, Bansal S, Girgis M, Iliuk A, Xu X, Fernandez JA, Griffin JH, Ballew EA, Unger K, Boerma M, Medhora M, Cheema AK Journal J Transl Med Abstract Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications ac (show more...)Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications across clinical research, including monitoring radiation exposure. A key limitation to their implementation is minimal standardization in EV isolation and analytical methods. Further, most urinary EV isolation protocols necessitate large volumes of sample. This study aimed to compare and optimize isolation and analytical methods for EVs from small volumes of urine. (hide) EV-METRIC 63% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Commercial method Protein markers EV: TSG101/ CD63/ CD81/ Alix/ Flotillin1/ EpCam/ ANXA5 non-EV: GM130 Proteomics no Show all info Study aim New methodological development/Biomarker/Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Urine Separation Method Commercial kit Other/ EVTrap Size-exclusion chromatography Resin type Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ Alix/ CD63/ TSG101/ EpCam/ ANXA5/ CD81 Detected contaminants GM130 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 115.9 EV concentration Yes EM EM-type Cryo-EM Image type Close-up

	EV210076	5/6	Rattus norvegicus	Urine	UF qEV SEC (non-commercial)	Hinzman CP	2022	63%
Study summary Full title An optimized method for the isolation of urinary extracellular vesicles for molecular phenotyping: detection of biomarkers for radiation exposure. All authors Hinzman CP, Jayatilake M, Bansal S, Fish BL, Li Y, Zhang Y, Bansal S, Girgis M, Iliuk A, Xu X, Fernandez JA, Griffin JH, Ballew EA, Unger K, Boerma M, Medhora M, Cheema AK Journal J Transl Med Abstract Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications ac (show more...)Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications across clinical research, including monitoring radiation exposure. A key limitation to their implementation is minimal standardization in EV isolation and analytical methods. Further, most urinary EV isolation protocols necessitate large volumes of sample. This study aimed to compare and optimize isolation and analytical methods for EVs from small volumes of urine. (hide) EV-METRIC 63% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin 13 Gy ionizing radiation Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Ultrafiltration Commercial method Size-exclusion chromatography (non-commercial) Protein markers EV: TSG101/ ANXA5/ CD63/ Alix/ CD81/ Flotillin1/ EpCam non-EV: GM130 Proteomics no Show all info Study aim New methodological development/Biomarker/Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Urine Separation Method Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Commercial kit qEV Size-exclusion chromatography Total column volume (mL) 3.5 Sample volume/column (mL) 0.15 Resin type Not Specified Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ EpCam/ ANXA5/ CD63/ TSG101/ Alix/ CD81 Detected contaminants GM130 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 144.4 EV concentration Yes EM EM-type Cryo-EM Image type Close-up

	EV210076	6/6	Rattus norvegicus	Urine	Other/ EVTrap	Hinzman CP	2022	63%
Study summary Full title An optimized method for the isolation of urinary extracellular vesicles for molecular phenotyping: detection of biomarkers for radiation exposure. All authors Hinzman CP, Jayatilake M, Bansal S, Fish BL, Li Y, Zhang Y, Bansal S, Girgis M, Iliuk A, Xu X, Fernandez JA, Griffin JH, Ballew EA, Unger K, Boerma M, Medhora M, Cheema AK Journal J Transl Med Abstract Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications ac (show more...)Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications across clinical research, including monitoring radiation exposure. A key limitation to their implementation is minimal standardization in EV isolation and analytical methods. Further, most urinary EV isolation protocols necessitate large volumes of sample. This study aimed to compare and optimize isolation and analytical methods for EVs from small volumes of urine. (hide) EV-METRIC 63% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin 13 Gy ionizing radiation Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Commercial method Protein markers EV: TSG101/ CD63/ CD81/ ANXA5/ Alix/ Flotillin1/ EpCam non-EV: GM130 Proteomics no Show all info Study aim New methodological development/Biomarker/Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Urine Separation Method Commercial kit Other/ EVTrap Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ Alix/ EpCam/ ANXA5/ TSG101/ CD63/ CD81 Detected contaminants GM130 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 125.2 EV concentration Yes EM EM-type Cryo-EM Image type Close-up

	EV220126	1/3	Sarcophilus harrisii	Serum	(d)(U)C UF qEV	Espejo C	2022	57%
Study summary Full title Cathelicidin-3 Associated With Serum Extracellular Vesicles Enables Early Diagnosis of a Transmissible Cancer. All authors Espejo C, Wilson R, Pye RJ, Ratcliffe JC, Ruiz-Aravena M, Willms E, Wolfe BW, Hamede R, Hill AF, Jones ME, Woods GM, Lyons AB Journal Front Immunol Abstract The identification of practical early diagnostic biomarkers is a cornerstone of improved prevention (show more...)The identification of practical early diagnostic biomarkers is a cornerstone of improved prevention and treatment of cancers. Such a case is devil facial tumor disease (DFTD), a highly lethal transmissible cancer afflicting virtually an entire species, the Tasmanian devil (). Despite a latent period that can exceed one year, to date DFTD diagnosis requires visual identification of tumor lesions. To enable earlier diagnosis, which is essential for the implementation of effective conservation strategies, we analyzed the extracellular vesicle (EV) proteome of 87 Tasmanian devil serum samples using data-independent acquisition mass spectrometry approaches. The antimicrobial peptide cathelicidin-3 (CATH3), released by innate immune cells, was enriched in serum EV samples of both devils with clinical DFTD (87.9% sensitivity and 94.1% specificity) and devils with latent infection (i.e., collected while overtly healthy, but 3-6 months before subsequent DFTD diagnosis/ 93.8% sensitivity and 94.1% specificity). Although high expression of antimicrobial peptides has been mostly related to inflammatory diseases, our results suggest that they can be also used as accurate cancer biomarkers, suggesting a mechanistic role in tumorous processes. This EV-based approach to biomarker discovery is directly applicable to improving understanding and diagnosis of a broad range of diseases in other species, and these findings directly enhance the capacity of conservation strategies to ensure the viability of the imperiled Tasmanian devil population. (hide) EV-METRIC 57% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Ultrafiltration qEV Protein markers EV: None non-EV: Albumin/ Lipoproteins Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Sarcophilus harrisii Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Proteomics database ProteomeXchange Consortium via Detected contaminants Albumin/ Lipoproteins Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 5 - 745 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.68E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220126	2/3	Sarcophilus harrisii	Serum	(d)(U)C UF qEV	Espejo C	2022	57%
Study summary Full title Cathelicidin-3 Associated With Serum Extracellular Vesicles Enables Early Diagnosis of a Transmissible Cancer. All authors Espejo C, Wilson R, Pye RJ, Ratcliffe JC, Ruiz-Aravena M, Willms E, Wolfe BW, Hamede R, Hill AF, Jones ME, Woods GM, Lyons AB Journal Front Immunol Abstract The identification of practical early diagnostic biomarkers is a cornerstone of improved prevention (show more...)The identification of practical early diagnostic biomarkers is a cornerstone of improved prevention and treatment of cancers. Such a case is devil facial tumor disease (DFTD), a highly lethal transmissible cancer afflicting virtually an entire species, the Tasmanian devil (). Despite a latent period that can exceed one year, to date DFTD diagnosis requires visual identification of tumor lesions. To enable earlier diagnosis, which is essential for the implementation of effective conservation strategies, we analyzed the extracellular vesicle (EV) proteome of 87 Tasmanian devil serum samples using data-independent acquisition mass spectrometry approaches. The antimicrobial peptide cathelicidin-3 (CATH3), released by innate immune cells, was enriched in serum EV samples of both devils with clinical DFTD (87.9% sensitivity and 94.1% specificity) and devils with latent infection (i.e., collected while overtly healthy, but 3-6 months before subsequent DFTD diagnosis/ 93.8% sensitivity and 94.1% specificity). Although high expression of antimicrobial peptides has been mostly related to inflammatory diseases, our results suggest that they can be also used as accurate cancer biomarkers, suggesting a mechanistic role in tumorous processes. This EV-based approach to biomarker discovery is directly applicable to improving understanding and diagnosis of a broad range of diseases in other species, and these findings directly enhance the capacity of conservation strategies to ensure the viability of the imperiled Tasmanian devil population. (hide) EV-METRIC 57% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Clinically diagnosed overt devil facial tumour disease Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Ultrafiltration qEV Protein markers EV: None non-EV: Albumin/ Lipoproteins Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Sarcophilus harrisii Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Proteomics database ProteomeXchange Consortium via Detected contaminants Albumin/ Lipoproteins Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 5 - 725 EV concentration Yes Particle yield particles per milliliter of starting sample: 6.08E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220126	3/3	Sarcophilus harrisii	Serum	(d)(U)C UF qEV	Espejo C	2022	57%
Study summary Full title Cathelicidin-3 Associated With Serum Extracellular Vesicles Enables Early Diagnosis of a Transmissible Cancer. All authors Espejo C, Wilson R, Pye RJ, Ratcliffe JC, Ruiz-Aravena M, Willms E, Wolfe BW, Hamede R, Hill AF, Jones ME, Woods GM, Lyons AB Journal Front Immunol Abstract The identification of practical early diagnostic biomarkers is a cornerstone of improved prevention (show more...)The identification of practical early diagnostic biomarkers is a cornerstone of improved prevention and treatment of cancers. Such a case is devil facial tumor disease (DFTD), a highly lethal transmissible cancer afflicting virtually an entire species, the Tasmanian devil (). Despite a latent period that can exceed one year, to date DFTD diagnosis requires visual identification of tumor lesions. To enable earlier diagnosis, which is essential for the implementation of effective conservation strategies, we analyzed the extracellular vesicle (EV) proteome of 87 Tasmanian devil serum samples using data-independent acquisition mass spectrometry approaches. The antimicrobial peptide cathelicidin-3 (CATH3), released by innate immune cells, was enriched in serum EV samples of both devils with clinical DFTD (87.9% sensitivity and 94.1% specificity) and devils with latent infection (i.e., collected while overtly healthy, but 3-6 months before subsequent DFTD diagnosis/ 93.8% sensitivity and 94.1% specificity). Although high expression of antimicrobial peptides has been mostly related to inflammatory diseases, our results suggest that they can be also used as accurate cancer biomarkers, suggesting a mechanistic role in tumorous processes. This EV-based approach to biomarker discovery is directly applicable to improving understanding and diagnosis of a broad range of diseases in other species, and these findings directly enhance the capacity of conservation strategies to ensure the viability of the imperiled Tasmanian devil population. (hide) EV-METRIC 57% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Latent devil facial tumour disease Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Ultrafiltration qEV Protein markers EV: None non-EV: Albumin/ Lipoproteins Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Sarcophilus harrisii Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Proteomics database ProteomeXchange Consortium via Detected contaminants Albumin/ Lipoproteins Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 15 - 745 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.24E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220089	4/14	Homo sapiens	HeLa-R19 cells	(d)(U)C DG	Susanne G. van der Grein	2022	57%
Study summary Full title The encephalomyocarditis virus Leader promotes the release of virions inside extracellular vesicles via the induction of secretory autophagy All authors Susanne G. van der Grein, Kyra A. Y. Defourny, Huib H. Rabouw, Soenita S. Goerdayal, Martijn J. C. van Herwijnen, Richard W. Wubbolts, Maarten Altelaar, Frank J. M. van Kuppeveld & Esther N. M. Nolte-‘t Hoen Journal Nature communications Abstract Naked viruses can escape host cells before the induction of lysis via release in extracellular vesic (show more...)Naked viruses can escape host cells before the induction of lysis via release in extracellular vesicles (EVs). These nanosized EVs cloak the secreted virus particles in a host-derived membrane, which alters virus-host interactions that affect infection efficiency and antiviral immunity. Currently, little is known about the viral and host factors regulating this form of virus release. Here, we assessed the role of the encephalomyocarditis virus (EMCV) Leader protein, a ‘viral security protein’ that subverts the host antiviral response. EV release upon infection with wildtype virus or a Leader-deficient mutant was characterized at the single particle level using high-resolution flow cytometry. Inactivation of the Leader abolished EV induction during infection and strongly reduced EV-enclosed virus release. We demonstrate that the Leader promotes the release of virions within EVs by stimulating a secretory arm of autophagy. This newly discovered role of the EMCV Leader adds to the variety of mechanisms via which this protein affects virus-host interactions. Moreover, these data provide first evidence for a crucial role of a non-structural viral protein in the non-lytic release of picornaviruses via packaging in EVs. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: None non-EV: None Proteomics yes EV density (g/ml) 1,10-1,04 Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa-R19 cells EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) 90 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100,000 Density gradient Type Continuous Lowest density fraction 5% Highest density fraction 45% Total gradient volume, incl. sample (mL) 11,3 Sample volume (mL) 0,3 Orientation Bottom-up Rotor type SW 40 Ti Speed (g) 192,000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing None Characterization: Protein analysis Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type In house modified BD influx jet in air sorter Hardware adjustment Please refer to: van der Vlist EJ, Nolte't Hoen EN, Stoorvogel W, Arkesteijn GJ, Wauben MH. Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry. Nat Protocol 2012/ 7(7):1311-1326. Calibration bead size 0,1 & 0,2 EV concentration Yes

	EV220089	8/14	Homo sapiens	HeLa-R19 cells	(d)(U)C DG	Susanne G. van der Grein	2022	57%
Study summary Full title The encephalomyocarditis virus Leader promotes the release of virions inside extracellular vesicles via the induction of secretory autophagy All authors Susanne G. van der Grein, Kyra A. Y. Defourny, Huib H. Rabouw, Soenita S. Goerdayal, Martijn J. C. van Herwijnen, Richard W. Wubbolts, Maarten Altelaar, Frank J. M. van Kuppeveld & Esther N. M. Nolte-‘t Hoen Journal Nature communications Abstract Naked viruses can escape host cells before the induction of lysis via release in extracellular vesic (show more...)Naked viruses can escape host cells before the induction of lysis via release in extracellular vesicles (EVs). These nanosized EVs cloak the secreted virus particles in a host-derived membrane, which alters virus-host interactions that affect infection efficiency and antiviral immunity. Currently, little is known about the viral and host factors regulating this form of virus release. Here, we assessed the role of the encephalomyocarditis virus (EMCV) Leader protein, a ‘viral security protein’ that subverts the host antiviral response. EV release upon infection with wildtype virus or a Leader-deficient mutant was characterized at the single particle level using high-resolution flow cytometry. Inactivation of the Leader abolished EV induction during infection and strongly reduced EV-enclosed virus release. We demonstrate that the Leader promotes the release of virions within EVs by stimulating a secretory arm of autophagy. This newly discovered role of the EMCV Leader adds to the variety of mechanisms via which this protein affects virus-host interactions. Moreover, these data provide first evidence for a crucial role of a non-structural viral protein in the non-lytic release of picornaviruses via packaging in EVs. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin EMCV-Wt virus infection Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: LC3 non-EV: None Proteomics yes EV density (g/ml) 1,10-1,04 Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa-R19 cells EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) 90 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 30 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 10,000 Density gradient Type Continuous Lowest density fraction 5% Highest density fraction 45% Total gradient volume, incl. sample (mL) 11,3 Sample volume (mL) 0,3 Orientation Bottom-up Rotor type SW 40 Ti Speed (g) 192,000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing None Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type In house modified BD influx jet in air sorter Hardware adjustment Please refer to: van der Vlist EJ, Nolte't Hoen EN, Stoorvogel W, Arkesteijn GJ, Wauben MH. Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry. Nat Protocol 2012/ 7(7):1311-1326. Calibration bead size 0,1 & 0,2 EV concentration Yes Extra information Mass spec database name will be included at a later stage. EVs were also analyzed for their viral content (based on virus titration).

	EV220089	9/14	Homo sapiens	HeLa-R19 cells	(d)(U)C DG	Susanne G. van der Grein	2022	57%
Study summary Full title The encephalomyocarditis virus Leader promotes the release of virions inside extracellular vesicles via the induction of secretory autophagy All authors Susanne G. van der Grein, Kyra A. Y. Defourny, Huib H. Rabouw, Soenita S. Goerdayal, Martijn J. C. van Herwijnen, Richard W. Wubbolts, Maarten Altelaar, Frank J. M. van Kuppeveld & Esther N. M. Nolte-‘t Hoen Journal Nature communications Abstract Naked viruses can escape host cells before the induction of lysis via release in extracellular vesic (show more...)Naked viruses can escape host cells before the induction of lysis via release in extracellular vesicles (EVs). These nanosized EVs cloak the secreted virus particles in a host-derived membrane, which alters virus-host interactions that affect infection efficiency and antiviral immunity. Currently, little is known about the viral and host factors regulating this form of virus release. Here, we assessed the role of the encephalomyocarditis virus (EMCV) Leader protein, a ‘viral security protein’ that subverts the host antiviral response. EV release upon infection with wildtype virus or a Leader-deficient mutant was characterized at the single particle level using high-resolution flow cytometry. Inactivation of the Leader abolished EV induction during infection and strongly reduced EV-enclosed virus release. We demonstrate that the Leader promotes the release of virions within EVs by stimulating a secretory arm of autophagy. This newly discovered role of the EMCV Leader adds to the variety of mechanisms via which this protein affects virus-host interactions. Moreover, these data provide first evidence for a crucial role of a non-structural viral protein in the non-lytic release of picornaviruses via packaging in EVs. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin EMCV-Wt virus infection Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: None non-EV: None Proteomics yes EV density (g/ml) 1,10-1,04 Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa-R19 cells EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) 90 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100,000 Density gradient Type Continuous Lowest density fraction 5% Highest density fraction 45% Total gradient volume, incl. sample (mL) 11,3 Sample volume (mL) 0,3 Orientation Bottom-up Rotor type SW 40 Ti Speed (g) 192,000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing None Characterization: Protein analysis Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type In house modified BD influx jet in air sorter Hardware adjustment Please refer to: van der Vlist EJ, Nolte't Hoen EN, Stoorvogel W, Arkesteijn GJ, Wauben MH. Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry. Nat Protocol 2012/ 7(7):1311-1326. Calibration bead size 0,1 & 0,2 EV concentration Yes

	EV220089	13/14	Homo sapiens	HeLa-R19 cells	(d)(U)C DG	Susanne G. van der Grein	2022	57%
Study summary Full title The encephalomyocarditis virus Leader promotes the release of virions inside extracellular vesicles via the induction of secretory autophagy All authors Susanne G. van der Grein, Kyra A. Y. Defourny, Huib H. Rabouw, Soenita S. Goerdayal, Martijn J. C. van Herwijnen, Richard W. Wubbolts, Maarten Altelaar, Frank J. M. van Kuppeveld & Esther N. M. Nolte-‘t Hoen Journal Nature communications Abstract Naked viruses can escape host cells before the induction of lysis via release in extracellular vesic (show more...)Naked viruses can escape host cells before the induction of lysis via release in extracellular vesicles (EVs). These nanosized EVs cloak the secreted virus particles in a host-derived membrane, which alters virus-host interactions that affect infection efficiency and antiviral immunity. Currently, little is known about the viral and host factors regulating this form of virus release. Here, we assessed the role of the encephalomyocarditis virus (EMCV) Leader protein, a ‘viral security protein’ that subverts the host antiviral response. EV release upon infection with wildtype virus or a Leader-deficient mutant was characterized at the single particle level using high-resolution flow cytometry. Inactivation of the Leader abolished EV induction during infection and strongly reduced EV-enclosed virus release. We demonstrate that the Leader promotes the release of virions within EVs by stimulating a secretory arm of autophagy. This newly discovered role of the EMCV Leader adds to the variety of mechanisms via which this protein affects virus-host interactions. Moreover, these data provide first evidence for a crucial role of a non-structural viral protein in the non-lytic release of picornaviruses via packaging in EVs. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin EMCV-L(Zn) virus infection Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: None non-EV: None Proteomics yes EV density (g/ml) 1,10-1,04 Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa-R19 cells EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) 90 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 30 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 10,000 Density gradient Type Continuous Lowest density fraction 5% Highest density fraction 45% Total gradient volume, incl. sample (mL) 11,3 Sample volume (mL) 0,3 Orientation Bottom-up Rotor type SW 40 Ti Speed (g) 192,000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing None Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type In house modified BD influx jet in air sorter Hardware adjustment Please refer to: van der Vlist EJ, Nolte't Hoen EN, Stoorvogel W, Arkesteijn GJ, Wauben MH. Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry. Nat Protocol 2012/ 7(7):1311-1326. Calibration bead size 0,1 & 0,2 EV concentration Yes Extra information Mass spec database name will be included at a later stage. EVs were also analyzed for their viral content (based on virus titration).

	EV220089	14/14	Homo sapiens	HeLa-R19 cells	(d)(U)C DG	Susanne G. van der Grein	2022	57%
Study summary Full title The encephalomyocarditis virus Leader promotes the release of virions inside extracellular vesicles via the induction of secretory autophagy All authors Susanne G. van der Grein, Kyra A. Y. Defourny, Huib H. Rabouw, Soenita S. Goerdayal, Martijn J. C. van Herwijnen, Richard W. Wubbolts, Maarten Altelaar, Frank J. M. van Kuppeveld & Esther N. M. Nolte-‘t Hoen Journal Nature communications Abstract Naked viruses can escape host cells before the induction of lysis via release in extracellular vesic (show more...)Naked viruses can escape host cells before the induction of lysis via release in extracellular vesicles (EVs). These nanosized EVs cloak the secreted virus particles in a host-derived membrane, which alters virus-host interactions that affect infection efficiency and antiviral immunity. Currently, little is known about the viral and host factors regulating this form of virus release. Here, we assessed the role of the encephalomyocarditis virus (EMCV) Leader protein, a ‘viral security protein’ that subverts the host antiviral response. EV release upon infection with wildtype virus or a Leader-deficient mutant was characterized at the single particle level using high-resolution flow cytometry. Inactivation of the Leader abolished EV induction during infection and strongly reduced EV-enclosed virus release. We demonstrate that the Leader promotes the release of virions within EVs by stimulating a secretory arm of autophagy. This newly discovered role of the EMCV Leader adds to the variety of mechanisms via which this protein affects virus-host interactions. Moreover, these data provide first evidence for a crucial role of a non-structural viral protein in the non-lytic release of picornaviruses via packaging in EVs. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin EMCV-L(Zn) virus infection Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: None non-EV: None Proteomics yes EV density (g/ml) 1,10-1,04 Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa-R19 cells EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) 90 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 30 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100,000 Density gradient Type Continuous Lowest density fraction 5% Highest density fraction 45% Total gradient volume, incl. sample (mL) 11,3 Sample volume (mL) 0,3 Orientation Bottom-up Rotor type SW 40 Ti Speed (g) 192,000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing None Characterization: Protein analysis Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type In house modified BD influx jet in air sorter Hardware adjustment Please refer to: van der Vlist EJ, Nolte't Hoen EN, Stoorvogel W, Arkesteijn GJ, Wauben MH. Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry. Nat Protocol 2012/ 7(7):1311-1326. Calibration bead size 0,1 & 0,2 EV concentration Yes

	EV210214	1/1	Homo sapiens	primary neutrophils	(d)(U)C UF SEC (non-commercial)	Bonifay, Amandine	2022	57%
Study summary Full title A new strategy to count and sort neutrophil‐derived extracellular vesicles: Validation in infectious disorders All authors Amandine Bonifay, Stéphane Robert, Belinda Champagne, Paul‐Rémi Petit, Aude Eugène, Corinne Chareyre, Anne‐Claire Duchez, Mélanie Vélier, Shirley Fritz, Loris Vallier, Romaric Lacroix, Françoise Dignat‐George Journal J Extracell Vesicles Abstract Newly recognized polymorphonuclear neutrophil (PMNs) functions include the ability to release subcel (show more...)Newly recognized polymorphonuclear neutrophil (PMNs) functions include the ability to release subcellular mediators such as neutrophil‐derived extracellular vesicles (NDEVs) involved in immune and thrombo‐inflammatory responses. Elevation of their plasmatic level has been reported in a variety of infectious and cardiovascular disorders, but the clinical use of this potential biomarker is hampered by methodological issues. Although flow cytometry (FCM) is currently used to detect NDEVs in the plasma of patients, an extensive characterization of NDEVs has never been done. Moreover, their detection remains challenging because of their small size and low antigen density. Therefore, the objective of the present study was first to establish a surface antigenic signature of NDEVs detectable by FCM and therefore to improve their detection in biological fluids by developing a strategy allowing to overcome their low fluorescent signal and reduce the background noise. By testing a large panel of 54 antibody specificities already reported to be positive on PMNs, we identified a profile of 15 membrane protein markers, including 4 (CD157, CD24, CD65 and CD66c) never described on NDEVs. Among them, CD15, CD66b and CD66c were identified as the most sensitive and specific markers to detect NDEVs by FCM. Using this antigenic signature, we developed a new strategy combining the three best antibodies in a cocktail and reducing the background noise by size exclusion chromatography (SEC). This strategy allowed a significant improvement in NDEVs enumeration in plasma from sepsis patients and made it feasible to efficiently sort NDEVs from COVID‐19 patients. Altogether, this work opens the door to a more valuable measurement of NDEVs as a potential biomarker in clinical practice. A similar strategy could also be applied to improve detection by FCM of other rare subpopulations of EVs generated by tissues with limited access, such as vascular endothelium, cancer cells or placenta. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles large extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD63/ GAPDH/ Integrin-beta3-subunit/ CD15/ CD66b/ CD44/ CD11c non-EV: Albumin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary neutrophils EV-harvesting Medium Serum-containing medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ GAPDH/ Integrin-beta3-subunit Detected contaminants Albumin Flow cytometry Type of Flow cytometry Cytoflex S Calibration bead size 0.1 0.16 0.22 0.24 0.3 0.5 0.9 Antibody details provided? Yes Detected EV-associated proteins CD15/ CD66b Not detected EV-associated proteins CD44/ CD11c Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Median Reported size (nm) 191 Particle analysis: flow cytometry Flow cytometer type Cytoflex S Hardware adjustment Calibration bead size 0.1 0.16 0.22 0.24 0.3 0.5 0.9 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV220431	4/4	Mus musculus	Serum	(d)(U)C IgG separation Filtration	Desai PP	2022	56%
Study summary Full title Combination of Small Extracellular Vesicle-Derived Annexin A2 Protein and mRNA as a Potential Predictive Biomarker for Chemotherapy Responsiveness in Aggressive Triple-Negative Breast Cancer. All authors Desai PP, Narra K, James JD, Jones HP, Tripathi AK, Vishwanatha JK Journal Cancers (Basel) Abstract Small extracellular vesicles (sEVs), mainly exosomes, are nanovesicles that shed from the membrane a (show more...)Small extracellular vesicles (sEVs), mainly exosomes, are nanovesicles that shed from the membrane as intraluminal vesicles of the multivesicular bodies, serve as vehicles that carry cargo influential in modulating the tumor microenvironment for the multi-step process of cancer metastasis. Annexin A2 (AnxA2), a calcium(Ca)-dependent phospholipid-binding protein, is among sEV cargoes. sEV-derived AnxA2 (sEV-AnxA2) protein is involved in the process of metastasis in triple-negative breast cancer (TNBC). The objective of the current study is to determine whether sEV-AnxA2 protein and/or mRNA could be a useful biomarkers to predict the responsiveness of chemotherapy in TNBC. Removal of Immunoglobulin G (IgG) from the serum as well as using the System Bioscience's ExoQuick Ultra kit resulted in efficient sEV isolation and detection of sEV-AnxA2 protein and mRNA compared to the ultracentrifugation method. The standardized method was applied to the twenty TNBC patient sera for sEV isolation. High levels of sEV-AnxA2 protein and/or mRNA were associated with stage 3 and above in TNBC. Four patients who responded to neoadjuvant chemotherapy had high expression of AnxA2 protein and/or mRNA in sEVs, while other four who did not respond to chemotherapy had low levels of AnxA2 protein and mRNA in sEVs. Our data suggest that the sEV-AnxA2 protein and mRNA could be a combined predictive biomarker for responsiveness to chemotherapy in aggressive TNBC. (hide) EV-METRIC 56% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Control condition Focus vesicles small extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation IgG separation Filtration Protein markers EV: CD9/ TSG101/ Annexin A2 non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 185000 Filtration steps 0.2 or 0.22 µm Other Name other separation method IgG separation Selected surface protein(s) Annexin A2 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins TSG101 Not detected EV-associated proteins Annexin A2/ CD9 Detected contaminants Calnexin/ GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 97-126 EV concentration Yes Particle yield Not determined

	EV210204	3/12	Homo sapiens	PANC-1	(d)(U)C Filtration Other/ EVTrap	Hinzman CP	2022	56%
Study summary Full title A multi-omics approach identifies pancreatic cancer cell extracellular vesicles as mediators of the unfolded protein response in normal pancreatic epithelial cells. All authors Hinzman CP, Singh B, Bansal S, Li Y, Iliuk A, Girgis M, Herremans KM, Trevino JG, Singh VK, Banerjee PP, Cheema AK Journal J Extracell Vesicles Abstract Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promotin (show more...)Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promoting cancer progression events, their precise effect on neighbouring normal cells is unknown. In this study, we investigated the impact of pancreatic cancer ductal adenocarcinoma (PDAC) derived EVs on recipient non-tumourigenic pancreatic normal epithelial cells upon internalization. We demonstrate that cEVs are readily internalized and induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in treated normal pancreatic epithelial cells within 24 h. We further show that PDAC cEVs increase cell proliferation, migration, and invasion and that these changes are regulated at least in part, by the UPR mediator DDIT3. Subsequently, these cells release several inflammatory cytokines. Leveraging a layered multi-omics approach, we analysed EV cargo from a panel of six PDAC and two normal pancreas cell lines, using multiple EV isolation methods. We found that cEVs were enriched for an array of biomolecules which can induce or regulate ER stress and the UPR, including palmitic acid, sphingomyelins, metabolic regulators of tRNA charging and proteins which regulate trafficking and degradation. We further show that palmitic acid, at doses relevant to those found in cEVs, is sufficient to induce ER stress in normal pancreas cells. These results suggest that cEV cargo packaging may be designed to disseminate proliferative and invasive characteristics upon internalization by distant recipient normal cells, hitherto unreported. This study is among the first to highlight a major role for PDAC cEVs to induce stress in treated normal pancreas cells that may modulate a systemic response leading to altered phenotypes. These findings highlight the importance of EVs in mediating disease aetiology and open potential areas of investigation toward understanding the role of cEV lipids in promoting cell transformation in the surrounding microenvironment. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Commercial method Protein markers EV: TSG101/ CD63/ CD81/ ANXA5/ Alix/ ICAM/ Flotillin1/ EpCAM non-EV: GM130 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PANC-1 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 120000 Filtration steps 0.22µm or 0.2µm Commercial kit Other/ EVTrap Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ Alix/ CD63/ TSG101/ ICAM/ EpCAM/ ANXA5/ CD81 Detected contaminants GM130 Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes

	EV210204	6/12	Homo sapiens	PPCL-68	(d)(U)C Filtration Other/ EVTrap	Hinzman CP	2022	56%
Study summary Full title A multi-omics approach identifies pancreatic cancer cell extracellular vesicles as mediators of the unfolded protein response in normal pancreatic epithelial cells. All authors Hinzman CP, Singh B, Bansal S, Li Y, Iliuk A, Girgis M, Herremans KM, Trevino JG, Singh VK, Banerjee PP, Cheema AK Journal J Extracell Vesicles Abstract Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promotin (show more...)Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promoting cancer progression events, their precise effect on neighbouring normal cells is unknown. In this study, we investigated the impact of pancreatic cancer ductal adenocarcinoma (PDAC) derived EVs on recipient non-tumourigenic pancreatic normal epithelial cells upon internalization. We demonstrate that cEVs are readily internalized and induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in treated normal pancreatic epithelial cells within 24 h. We further show that PDAC cEVs increase cell proliferation, migration, and invasion and that these changes are regulated at least in part, by the UPR mediator DDIT3. Subsequently, these cells release several inflammatory cytokines. Leveraging a layered multi-omics approach, we analysed EV cargo from a panel of six PDAC and two normal pancreas cell lines, using multiple EV isolation methods. We found that cEVs were enriched for an array of biomolecules which can induce or regulate ER stress and the UPR, including palmitic acid, sphingomyelins, metabolic regulators of tRNA charging and proteins which regulate trafficking and degradation. We further show that palmitic acid, at doses relevant to those found in cEVs, is sufficient to induce ER stress in normal pancreas cells. These results suggest that cEV cargo packaging may be designed to disseminate proliferative and invasive characteristics upon internalization by distant recipient normal cells, hitherto unreported. This study is among the first to highlight a major role for PDAC cEVs to induce stress in treated normal pancreas cells that may modulate a systemic response leading to altered phenotypes. These findings highlight the importance of EVs in mediating disease aetiology and open potential areas of investigation toward understanding the role of cEV lipids in promoting cell transformation in the surrounding microenvironment. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Commercial method Protein markers EV: TSG101/ CD63/ CD81/ ANXA5/ Alix/ ICAM/ Flotillin1/ EpCAM non-EV: GM130 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PPCL-68 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 120000 Filtration steps 0.22µm or 0.2µm Commercial kit Other/ EVTrap Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ Alix/ CD63/ TSG101/ ICAM/ EpCAM/ ANXA5/ CD81 Detected contaminants GM130 Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes

	EV210204	9/12	Homo sapiens	hTERT-HPNE	(d)(U)C Filtration Other/ EVTrap	Hinzman CP	2022	56%
Study summary Full title A multi-omics approach identifies pancreatic cancer cell extracellular vesicles as mediators of the unfolded protein response in normal pancreatic epithelial cells. All authors Hinzman CP, Singh B, Bansal S, Li Y, Iliuk A, Girgis M, Herremans KM, Trevino JG, Singh VK, Banerjee PP, Cheema AK Journal J Extracell Vesicles Abstract Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promotin (show more...)Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promoting cancer progression events, their precise effect on neighbouring normal cells is unknown. In this study, we investigated the impact of pancreatic cancer ductal adenocarcinoma (PDAC) derived EVs on recipient non-tumourigenic pancreatic normal epithelial cells upon internalization. We demonstrate that cEVs are readily internalized and induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in treated normal pancreatic epithelial cells within 24 h. We further show that PDAC cEVs increase cell proliferation, migration, and invasion and that these changes are regulated at least in part, by the UPR mediator DDIT3. Subsequently, these cells release several inflammatory cytokines. Leveraging a layered multi-omics approach, we analysed EV cargo from a panel of six PDAC and two normal pancreas cell lines, using multiple EV isolation methods. We found that cEVs were enriched for an array of biomolecules which can induce or regulate ER stress and the UPR, including palmitic acid, sphingomyelins, metabolic regulators of tRNA charging and proteins which regulate trafficking and degradation. We further show that palmitic acid, at doses relevant to those found in cEVs, is sufficient to induce ER stress in normal pancreas cells. These results suggest that cEV cargo packaging may be designed to disseminate proliferative and invasive characteristics upon internalization by distant recipient normal cells, hitherto unreported. This study is among the first to highlight a major role for PDAC cEVs to induce stress in treated normal pancreas cells that may modulate a systemic response leading to altered phenotypes. These findings highlight the importance of EVs in mediating disease aetiology and open potential areas of investigation toward understanding the role of cEV lipids in promoting cell transformation in the surrounding microenvironment. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Commercial method Protein markers EV: TSG101/ CD63/ CD81/ ANXA5/ Alix/ ICAM/ Flotillin1/ EpCAM non-EV: GM130 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hTERT-HPNE EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 120000 Filtration steps 0.22µm or 0.2µm Commercial kit Other/ EVTrap Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ Alix/ CD63/ TSG101/ ICAM/ EpCAM/ ANXA5/ CD81 Detected contaminants GM130 Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes

	EV210204	12/12	Homo sapiens	HPDE-H6c7	(d)(U)C Filtration Other/ EVTrap	Hinzman CP	2022	56%
Study summary Full title A multi-omics approach identifies pancreatic cancer cell extracellular vesicles as mediators of the unfolded protein response in normal pancreatic epithelial cells. All authors Hinzman CP, Singh B, Bansal S, Li Y, Iliuk A, Girgis M, Herremans KM, Trevino JG, Singh VK, Banerjee PP, Cheema AK Journal J Extracell Vesicles Abstract Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promotin (show more...)Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promoting cancer progression events, their precise effect on neighbouring normal cells is unknown. In this study, we investigated the impact of pancreatic cancer ductal adenocarcinoma (PDAC) derived EVs on recipient non-tumourigenic pancreatic normal epithelial cells upon internalization. We demonstrate that cEVs are readily internalized and induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in treated normal pancreatic epithelial cells within 24 h. We further show that PDAC cEVs increase cell proliferation, migration, and invasion and that these changes are regulated at least in part, by the UPR mediator DDIT3. Subsequently, these cells release several inflammatory cytokines. Leveraging a layered multi-omics approach, we analysed EV cargo from a panel of six PDAC and two normal pancreas cell lines, using multiple EV isolation methods. We found that cEVs were enriched for an array of biomolecules which can induce or regulate ER stress and the UPR, including palmitic acid, sphingomyelins, metabolic regulators of tRNA charging and proteins which regulate trafficking and degradation. We further show that palmitic acid, at doses relevant to those found in cEVs, is sufficient to induce ER stress in normal pancreas cells. These results suggest that cEV cargo packaging may be designed to disseminate proliferative and invasive characteristics upon internalization by distant recipient normal cells, hitherto unreported. This study is among the first to highlight a major role for PDAC cEVs to induce stress in treated normal pancreas cells that may modulate a systemic response leading to altered phenotypes. These findings highlight the importance of EVs in mediating disease aetiology and open potential areas of investigation toward understanding the role of cEV lipids in promoting cell transformation in the surrounding microenvironment. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Commercial method Protein markers EV: TSG101/ CD63/ CD81/ ANXA5/ Alix/ ICAM/ Flotillin1/ EpCAM non-EV: GM130 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HPDE-H6c7 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 120000 Filtration steps 0.22µm or 0.2µm Commercial kit Other/ EVTrap Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ Alix/ CD63/ TSG101/ ICAM/ EpCAM/ ANXA5/ CD81 Detected contaminants GM130 Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes

	EV220307	3/3	Homo sapiens	CENH	(d)(U)C	Berry F	2022	56%
Study summary Full title Proviral role of human respiratory epithelial cell-derived small extracellular vesicles in SARS-CoV-2 infection. All authors Berry F, Morin-Dewaele M, Majidipur A, Jamet T, Bartier S, Ignjatovic E, Toniutti D, Gaspar Lopes J, Soyeux-Porte P, Maillé P, Saldana C, Brillet R, Ahnou N, Softic L, Couturaud B, Huet É, Ahmed-Belkacem A, Fourati S, Louis B, Coste A, Béquignon É, de la Taille A, Destouches D, Vacherot F, Pawlotsky JM, Firlej V, Bruscella P Journal J Extracell Vesicles Abstract Small Extracellular Vesicles (sEVs) are 50-200 nm in diameter vesicles delimited by a lipid bilaye (show more...)Small Extracellular Vesicles (sEVs) are 50-200 nm in diameter vesicles delimited by a lipid bilayer, formed within the endosomal network or derived from the plasma membrane. They are secreted in various biological fluids, including airway nasal mucus. The goal of this work was to understand the role of sEVs present in the mucus (mu-sEVs) produced by human nasal epithelial cells (HNECs) in SARS-CoV-2 infection. We show that uninfected HNECs produce mu-sEVs containing SARS-CoV-2 receptor ACE2 and activated protease TMPRSS2. mu-sEVs cleave prefusion viral Spike proteins at the S1/S2 boundary, resulting in higher proportions of prefusion S proteins exposing their receptor binding domain in an 'open' conformation, thereby facilitating receptor binding at the cell surface. We show that the role of nasal mu-sEVs is to complete prefusion Spike priming performed by intracellular furin during viral egress from infected cells. This effect is mediated by vesicular TMPRSS2 activity, rendering SARS-CoV-2 virions prone to entry into target cells using the 'early', TMPRSS2-dependent pathway instead of the 'late', cathepsin-dependent route. These results indicate that prefusion Spike priming by mu-sEVs in the nasal cavity plays a role in viral tropism. They also show that nasal mucus does not protect from SARS-CoV-2 infection, but instead facilitates it. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD9/ TSG101/ CD81/ ACE2/ TMPRSS2 non-EV: Calreticulin/ cytochrome C Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells CENH Cell viability (%) 100 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type Type 90 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 1 Wash: time (min) 90 Wash: Rotor Type Type 90 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ TSG101/ ACE2/ TMPRSS2 Not detected EV-associated proteins CD81 Not detected contaminants Calreticulin/ cytochrome C Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 156 EV concentration Yes Particle yield yes, as number of particles per insert: 3.30E+10 EM EM-type Transmission-EM Image type Wide-field

	EV220119	3/14	Schistosoma mansoni	whole parasite culture	(d)(U)C DG	Kuipers, Marije	2022	56%
Study summary Full title Optimized Protocol for the Isolation of Extracellular Vesicles from the Parasitic Worm Schistosoma mansoni with Improved Purity, Concentration, and Yield All authors Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen Journal J immunol Res Abstract In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide) EV-METRIC 56% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type whole parasite culture Sample origin adult worm Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: S. mansoni TSP-2 non-EV: None Proteomics no EV density (g/ml) 1.07-1.15 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Schistosoma mansoni Sample Type whole parasite culture Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 96,808 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 10% Highest density fraction 60% (58,7% with sample) Total gradient volume, incl. sample (mL) 11.97 Sample volume (mL) 0.07 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 169,044 Duration (min) >960 Fraction volume (mL) 0.997 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins S. mansoni TSP-2 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220119	4/14	Schistosoma mansoni	whole parasite culture	(d)(U)C DG	Kuipers, Marije	2022	56%
Study summary Full title Optimized Protocol for the Isolation of Extracellular Vesicles from the Parasitic Worm Schistosoma mansoni with Improved Purity, Concentration, and Yield All authors Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen Journal J immunol Res Abstract In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide) EV-METRIC 56% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type whole parasite culture Sample origin adult worm Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: S. mansoni TSP-2 non-EV: None Proteomics no EV density (g/ml) 1.07-1.21 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Schistosoma mansoni Sample Type whole parasite culture Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 96,808 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 10% Highest density fraction 60% (56.4 % with sample) Total gradient volume, incl. sample (mL) 4.25 Sample volume (mL) 0.07 Orientation Bottom-up Rotor type SW 55 Ti Speed (g) 169,639 Duration (min) >960 Fraction volume (mL) 0.354 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins S. mansoni TSP-2 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220119	5/14	Schistosoma mansoni	whole parasite culture	(d)(U)C DG	Kuipers, Marije	2022	56%
Study summary Full title Optimized Protocol for the Isolation of Extracellular Vesicles from the Parasitic Worm Schistosoma mansoni with Improved Purity, Concentration, and Yield All authors Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen Journal J immunol Res Abstract In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide) EV-METRIC 56% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type whole parasite culture Sample origin adult worm Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: S. mansoni TSP-2 non-EV: None Proteomics no EV density (g/ml) 1.07-1.21 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Schistosoma mansoni Sample Type whole parasite culture Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 96,808 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 10% Highest density fraction 60% (51.8 % with sample) Total gradient volume, incl. sample (mL) 1.742 Sample volume (mL) 0.07 Orientation Bottom-up Rotor type TLS-55 Speed (g) 166,180 Duration (min) 120 Fraction volume (mL) 0.145 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins S. mansoni TSP-2 Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Cryo-EM Image type Wide-field

	EV220119	6/14	Schistosoma mansoni	whole parasite culture	(d)(U)C DG	Kuipers, Marije	2022	56%
Study summary Full title Optimized Protocol for the Isolation of Extracellular Vesicles from the Parasitic Worm Schistosoma mansoni with Improved Purity, Concentration, and Yield All authors Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen Journal J immunol Res Abstract In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide) EV-METRIC 56% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type whole parasite culture Sample origin adult worm Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: S. mansoni TSP-2 non-EV: None Proteomics no EV density (g/ml) 1.07-1.19 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Schistosoma mansoni Sample Type whole parasite culture Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 96,808 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 10% Highest density fraction 60% Total gradient volume, incl. sample (mL) 1.742 Sample volume (mL) 0.07 Orientation Top-down Rotor type TLS-55 Speed (g) 166,180 Duration (min) 120 Fraction volume (mL) 0.145 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins S. mansoni TSP-2 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220119	7/14	Schistosoma mansoni	whole parasite culture	(d)(U)C DG	Kuipers, Marije	2022	56%
Study summary Full title Optimized Protocol for the Isolation of Extracellular Vesicles from the Parasitic Worm Schistosoma mansoni with Improved Purity, Concentration, and Yield All authors Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen Journal J immunol Res Abstract In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide) EV-METRIC 56% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type whole parasite culture Sample origin adult worm Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: S. mansoni TSP-2 non-EV: None Proteomics no EV density (g/ml) 1.25-1.11 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Schistosoma mansoni Sample Type whole parasite culture Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 96,808 Density gradient Type Discontinuous Number of initial discontinuous layers 15 Lowest density fraction 0.4M Highest density fraction 2.5M (2.05M with sample) Total gradient volume, incl. sample (mL) 1.86 Sample volume (mL) 0.07 Orientation Bottom-up Rotor type TLS-55 Speed (g) 166,180 Duration (min) 120 Fraction volume (mL) 0.155 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins S. mansoni TSP-2 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220119	8/14	Schistosoma mansoni	whole parasite culture	(d)(U)C DG	Kuipers, Marije	2022	56%
Study summary Full title Optimized Protocol for the Isolation of Extracellular Vesicles from the Parasitic Worm Schistosoma mansoni with Improved Purity, Concentration, and Yield All authors Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen Journal J immunol Res Abstract In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide) EV-METRIC 56% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type whole parasite culture Sample origin adult worm Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: S. mansoni TSP-2 non-EV: None Proteomics no EV density (g/ml) 1.20-1.12 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Schistosoma mansoni Sample Type whole parasite culture Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 96,808 Density gradient Type Discontinuous Number of initial discontinuous layers 15 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 1.86 Sample volume (mL) 0.07 Orientation Top-down Rotor type TLS-55 Speed (g) 166,180 Duration (min) 120 Fraction volume (mL) 0.155 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins S. mansoni TSP-2 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220119	9/14	Schistosoma mansoni	whole parasite culture	(d)(U)C DG	Kuipers, Marije	2022	56%
Study summary Full title Optimized Protocol for the Isolation of Extracellular Vesicles from the Parasitic Worm Schistosoma mansoni with Improved Purity, Concentration, and Yield All authors Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen Journal J immunol Res Abstract In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide) EV-METRIC 56% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type whole parasite culture Sample origin adult worm Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: S. mansoni TSP-2 non-EV: None Proteomics no EV density (g/ml) 1.17-1.12 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Schistosoma mansoni Sample Type whole parasite culture Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 96,808 Density gradient Type Discontinuous Number of initial discontinuous layers 15 Lowest density fraction 0.4M Highest density fraction 2.5M (2.05M with sample) Total gradient volume, incl. sample (mL) 1.86 Sample volume (mL) 0.07 Orientation Bottom-up Rotor type TLS-55 Speed (g) 166,180 Duration (min) 810 Fraction volume (mL) 0.155 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins S. mansoni TSP-2 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220119	11/14	Schistosoma mansoni	whole parasite culture	(d)(U)C DG	Kuipers, Marije	2022	56%
Study summary Full title Optimized Protocol for the Isolation of Extracellular Vesicles from the Parasitic Worm Schistosoma mansoni with Improved Purity, Concentration, and Yield All authors Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen Journal J immunol Res Abstract In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide) EV-METRIC 56% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type whole parasite culture Sample origin schistosomula Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: S. mansoni TSP-2 non-EV: None Proteomics no EV density (g/ml) 1.07-1.21 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Schistosoma mansoni Sample Type whole parasite culture Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 96,808 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 10% Highest density fraction 60% (51.8 % with sample) Total gradient volume, incl. sample (mL) 1.742 Sample volume (mL) 0.07 Orientation Bottom-up Rotor type TLS-55 Speed (g) 166,180 Duration (min) 120 Fraction volume (mL) 0.145 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins S. mansoni TSP-2 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220119	12/14	Schistosoma mansoni	whole parasite culture	(d)(U)C DG	Kuipers, Marije	2022	56%
Study summary Full title Optimized Protocol for the Isolation of Extracellular Vesicles from the Parasitic Worm Schistosoma mansoni with Improved Purity, Concentration, and Yield All authors Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen Journal J immunol Res Abstract In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide) EV-METRIC 56% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type whole parasite culture Sample origin schistosomula Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: S. mansoni TSP-2 non-EV: None Proteomics no EV density (g/ml) 1.07-1.19 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Schistosoma mansoni Sample Type whole parasite culture Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 96,808 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 10% Highest density fraction 60% Total gradient volume, incl. sample (mL) 1.742 Sample volume (mL) 0.07 Orientation Top-down Rotor type TLS-55 Speed (g) 166,180 Duration (min) 120 Fraction volume (mL) 0.145 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins S. mansoni TSP-2 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220119	13/14	Schistosoma mansoni	whole parasite culture	(d)(U)C DG	Kuipers, Marije	2022	56%
Study summary Full title Optimized Protocol for the Isolation of Extracellular Vesicles from the Parasitic Worm Schistosoma mansoni with Improved Purity, Concentration, and Yield All authors Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen Journal J immunol Res Abstract In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide) EV-METRIC 56% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type whole parasite culture Sample origin schistosomula Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: S. mansoni TSP-2 non-EV: None Proteomics no EV density (g/ml) 1.26-1.25 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Schistosoma mansoni Sample Type whole parasite culture Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 96,808 Density gradient Type Discontinuous Number of initial discontinuous layers 15 Lowest density fraction 0.4M Highest density fraction 2.5M (2.05M with sample) Total gradient volume, incl. sample (mL) 1.86 Sample volume (mL) 0.07 Orientation Bottom-up Rotor type TLS-55 Speed (g) 166,180 Duration (min) 810 Fraction volume (mL) 0.155 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins S. mansoni TSP-2 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220119	14/14	Schistosoma mansoni	whole parasite culture	(d)(U)C DG	Kuipers, Marije	2022	56%
Study summary Full title Optimized Protocol for the Isolation of Extracellular Vesicles from the Parasitic Worm Schistosoma mansoni with Improved Purity, Concentration, and Yield All authors Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen Journal J immunol Res Abstract In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide) EV-METRIC 56% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type whole parasite culture Sample origin schistosomula Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: S. mansoni TSP-2 non-EV: None Proteomics no EV density (g/ml) 1.28-1.21 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Schistosoma mansoni Sample Type whole parasite culture Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 96,808 Density gradient Type Discontinuous Number of initial discontinuous layers 15 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 1.86 Sample volume (mL) 0.07 Orientation Top-down Rotor type TLS-55 Speed (g) 166,180 Duration (min) 810 Fraction volume (mL) 0.155 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins S. mansoni TSP-2 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220069	1/11	Homo sapiens	MHCC97L	(d)(U)C	Tey, Sze Kong	2022	56%
Study summary Full title Liver cancer cells with nuclear MET overexpression release translation regulatory protein-enriched extracellular vesicles exhibit metastasis promoting activity All authors Sze Keong Tey, Samuel Wan Ki Wong, Cherlie Lot Sum Yeung, Jason Ying Ki Li, Xiaowen Mao, Clive Yik Sham Chung, Judy Wai Ping Yam Journal Journal of Extracellular Biology Abstract MET receptor tyrosine kinase is a cell surface receptor that plays important role in embryonic devel (show more...)MET receptor tyrosine kinase is a cell surface receptor that plays important role in embryonic development and tissue regeneration. Aberrant MET activation has been widely reported in different human cancers, making MET an attractive therapeutic target. The presence of truncated MET within the nucleus (nMET) with potential novel functions poses a great challenge to the current therapeutic strategies against MET surface receptor. Previous work has demonstrated the promoting effect of nMET in aggressive properties of hepatocellular carcinoma (HCC) cells by activating TAK1/NF-κB signalling pathway. Herein, we report the role of nMET in modulating tumour microenvironment and tumour metastasis mediated by extracellular vesicles (EVs). EVs released by nMET overexpressing cells enhanced cell motility and provoked metastasis. Proteomic profiling revealed the enrichment of translational regulatory proteins in EVs derived from nMET overexpressing cells. These proteins include eukaryotic initiation factor (EIF), ribosomal protein small subunit (RPS) and ribosomal protein larger subunit (RPL) gene families. Knockdown of EIF3I, RPS3A and RPL10 diminished the promoting effect of EVs in cell migration invasiveness and metastasis. In conclusion, the findings reveal an unrecognized capacity of nMET to augment HCC through the release of EVs with oncogenic effect. Targeting these translation-related proteins may serve as an alternative treatment for patients with nMET overexpression. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD9/ TSG101 non-EV: GM130/ tubulin-alpha/ Calreticulin/ Albumin/ Argonaute2/ PMP70/ Prohibitin/ Tamm-Horsfall protein Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MHCC97L EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 70 Wash: time (min) 70 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ TSG101 Not detected contaminants GM130/ tubulin-alpha Proteomics database ProteomeXchange Detected contaminants Calreticulin Not detected contaminants Albumin/ Argonaute2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 156.5 EM EM-type Immuno-EM EM protein CD63 Image type Close-up

	EV220069	2/11	Homo sapiens	MHCC97L	(d)(U)C	Tey, Sze Kong	2022	56%
Study summary Full title Liver cancer cells with nuclear MET overexpression release translation regulatory protein-enriched extracellular vesicles exhibit metastasis promoting activity All authors Sze Keong Tey, Samuel Wan Ki Wong, Cherlie Lot Sum Yeung, Jason Ying Ki Li, Xiaowen Mao, Clive Yik Sham Chung, Judy Wai Ping Yam Journal Journal of Extracellular Biology Abstract MET receptor tyrosine kinase is a cell surface receptor that plays important role in embryonic devel (show more...)MET receptor tyrosine kinase is a cell surface receptor that plays important role in embryonic development and tissue regeneration. Aberrant MET activation has been widely reported in different human cancers, making MET an attractive therapeutic target. The presence of truncated MET within the nucleus (nMET) with potential novel functions poses a great challenge to the current therapeutic strategies against MET surface receptor. Previous work has demonstrated the promoting effect of nMET in aggressive properties of hepatocellular carcinoma (HCC) cells by activating TAK1/NF-κB signalling pathway. Herein, we report the role of nMET in modulating tumour microenvironment and tumour metastasis mediated by extracellular vesicles (EVs). EVs released by nMET overexpressing cells enhanced cell motility and provoked metastasis. Proteomic profiling revealed the enrichment of translational regulatory proteins in EVs derived from nMET overexpressing cells. These proteins include eukaryotic initiation factor (EIF), ribosomal protein small subunit (RPS) and ribosomal protein larger subunit (RPL) gene families. Knockdown of EIF3I, RPS3A and RPL10 diminished the promoting effect of EVs in cell migration invasiveness and metastasis. In conclusion, the findings reveal an unrecognized capacity of nMET to augment HCC through the release of EVs with oncogenic effect. Targeting these translation-related proteins may serve as an alternative treatment for patients with nMET overexpression. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin nMet overexpressing Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD9/ TSG101 non-EV: GM130/ tubulin-alpha/ Calreticulin/ Albumin/ Argonaute2/ PMP70/ Prohibitin/ Tamm-Horsfall protein Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MHCC97L EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 70 Wash: time (min) 70 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ TSG101 Not detected contaminants GM130/ tubulin-alpha Proteomics database ProteomeXchange Detected contaminants Calreticulin Not detected contaminants Albumin/ Argonaute2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 155.1 EM EM-type Immuno-EM EM protein CD63 Image type Close-up

	EV220040	1/3	Homo sapiens	SW620	(d)(U)C	Santos MF	2022	56%
Study summary Full title Anti-Human CD9 Fab Fragment Antibody Blocks the Extracellular Vesicle-Mediated Increase in Malignancy of Colon Cancer Cells. All authors Santos MF, Rappa G, Fontana S, Karbanová J, Aalam F, Tai D, Li Z, Pucci M, Alessandro R, Morimoto C, Corbeil D, Lorico A Journal Cells Abstract Intercellular communication between cancer cells themselves or with healthy cells in the tumor micro (show more...)Intercellular communication between cancer cells themselves or with healthy cells in the tumor microenvironment and/or pre-metastatic sites plays an important role in cancer progression and metastasis. In addition to ligand-receptor signaling complexes, extracellular vesicles (EVs) are emerging as novel mediators of intercellular communication both in tissue homeostasis and in diseases such as cancer. EV-mediated transfer of molecular activities impacting morphological features and cell motility from highly metastatic SW620 cells to non-metastatic SW480 cells is a good in vitro example to illustrate the increased malignancy of colorectal cancer leading to its transformation and aggressive behavior. In an attempt to intercept the intercellular communication promoted by EVs, we recently developed a monovalent Fab fragment antibody directed against human CD9 tetraspanin and showed its effectiveness in blocking the internalization of melanoma cell-derived EVs and the nuclear transfer of their cargo proteins into recipient cells. Here, we employed the SW480/SW620 model to investigate the anti-cancer potential of the anti-CD9 Fab antibody. We first demonstrated that most EVs derived from SW620 cells contain CD9, making them potential targets. We then found that the anti-CD9 Fab antibody, but not the corresponding divalent antibody, prevented internalization of EVs from SW620 cells into SW480 cells, thereby inhibiting their phenotypic transformation, i.e., the change from a mesenchymal-like morphology to a rounded amoeboid-like shape with membrane blebbing, and thus preventing increased cell migration. Intercepting EV-mediated intercellular communication in the tumor niche with an anti-CD9 Fab antibody, combined with direct targeting of cancer cells, could lead to the development of new anti-cancer therapeutic strategies. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD9/ CD63/ CD81 non-EV: Calnexin Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SW620 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 200000 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ CD63/ CD81 Not detected contaminants Calnexin Other 1 High resolution STORM micrsocopy Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 148 EV concentration Yes Particle yield particles per milliliter of starting sample: 6.90E+10 Other particle analysis name(1) High resolution STORM microscopy EV-concentration No

	EV220040	2/3	Homo sapiens	SW620	(d)(U)C	Santos MF	2022	56%
Study summary Full title Anti-Human CD9 Fab Fragment Antibody Blocks the Extracellular Vesicle-Mediated Increase in Malignancy of Colon Cancer Cells. All authors Santos MF, Rappa G, Fontana S, Karbanová J, Aalam F, Tai D, Li Z, Pucci M, Alessandro R, Morimoto C, Corbeil D, Lorico A Journal Cells Abstract Intercellular communication between cancer cells themselves or with healthy cells in the tumor micro (show more...)Intercellular communication between cancer cells themselves or with healthy cells in the tumor microenvironment and/or pre-metastatic sites plays an important role in cancer progression and metastasis. In addition to ligand-receptor signaling complexes, extracellular vesicles (EVs) are emerging as novel mediators of intercellular communication both in tissue homeostasis and in diseases such as cancer. EV-mediated transfer of molecular activities impacting morphological features and cell motility from highly metastatic SW620 cells to non-metastatic SW480 cells is a good in vitro example to illustrate the increased malignancy of colorectal cancer leading to its transformation and aggressive behavior. In an attempt to intercept the intercellular communication promoted by EVs, we recently developed a monovalent Fab fragment antibody directed against human CD9 tetraspanin and showed its effectiveness in blocking the internalization of melanoma cell-derived EVs and the nuclear transfer of their cargo proteins into recipient cells. Here, we employed the SW480/SW620 model to investigate the anti-cancer potential of the anti-CD9 Fab antibody. We first demonstrated that most EVs derived from SW620 cells contain CD9, making them potential targets. We then found that the anti-CD9 Fab antibody, but not the corresponding divalent antibody, prevented internalization of EVs from SW620 cells into SW480 cells, thereby inhibiting their phenotypic transformation, i.e., the change from a mesenchymal-like morphology to a rounded amoeboid-like shape with membrane blebbing, and thus preventing increased cell migration. Intercepting EV-mediated intercellular communication in the tumor niche with an anti-CD9 Fab antibody, combined with direct targeting of cancer cells, could lead to the development of new anti-cancer therapeutic strategies. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin CD9-GFP exression Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD9/ CD63/ CD81 non-EV: Calnexin Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SW620 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 200000 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ CD63/ CD81 Not detected contaminants Calnexin Other 1 High resolution STORM microscopy Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 157 EV concentration Yes Particle yield particles per milliliter of starting sample: 7.60E+10 Other particle analysis name(1) High resolution STORM microscopy EV-concentration No

	EV210345	2/17	Homo sapiens	Expi293F	(d)(U)C	Osteikoetxea X	2022	56%
Study summary Full title Engineered Cas9 extracellular vesicles as a novel gene editing tool. All authors Osteikoetxea X, Silva A, Lázaro-Ibáñez E, Salmond N, Shatnyeva O, Stein J, Schick J, Wren S, Lindgren J, Firth M, Madsen A, Mayr LM, Overman R, Davies R, Dekker N Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic app (show more...)Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin CD81-CIBN-CRY2-Cas9 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD63/ CD81/ Flotillin1/ TSG101/ syntenin-1/ B-actin non-EV: calnexin Proteomics no Show all info Study aim Function/Drug delivery Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 95.4 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 38 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Size-exclusion chromatography Resin type Characterization: Protein analysis Protein Concentration Method Nanodrop Protein Yield (µg) number of particles per million cells Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD63/ CD81/ Flotillin1/ TSG101/ syntenin-1/ B-actin/ spCas9 Detected contaminants calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 120 EV concentration Yes Particle yield number of particles per million cells: 5.00e+4

	EV210345	4/17	Homo sapiens	Expi293F	(d)(U)C	Osteikoetxea X	2022	56%
Study summary Full title Engineered Cas9 extracellular vesicles as a novel gene editing tool. All authors Osteikoetxea X, Silva A, Lázaro-Ibáñez E, Salmond N, Shatnyeva O, Stein J, Schick J, Wren S, Lindgren J, Firth M, Madsen A, Mayr LM, Overman R, Davies R, Dekker N Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic app (show more...)Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Mys-CIBN-CRY2-Cas9 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD63/ CD81/ Flotillin1/ TSG101/ B-actin/ syntenin-1 non-EV: Calnexin Proteomics no Show all info Study aim Function/Drug delivery Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 95.4 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 38 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Size-exclusion chromatography Resin type Characterization: Protein analysis Protein Concentration Method Nanodrop Protein Yield (µg) number of particles per million cells Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD63/ CD81/ Flotillin1/ TSG101/ B-actin/ syntenin-1/ spCas9 Detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 120 EV concentration Yes Particle yield number of particles per million cells: 4.00e+4

	EV210345	16/17	Homo sapiens	Expi293F	(d)(U)C	Osteikoetxea X	2022	56%
Study summary Full title Engineered Cas9 extracellular vesicles as a novel gene editing tool. All authors Osteikoetxea X, Silva A, Lázaro-Ibáñez E, Salmond N, Shatnyeva O, Stein J, Schick J, Wren S, Lindgren J, Firth M, Madsen A, Mayr LM, Overman R, Davies R, Dekker N Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic app (show more...)Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin CD9-FKBP-FRB-Cas9 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD63/ CD81/ Flotillin1/ TSG101/ B-actin/ syntenin-1 non-EV: Calnexin Proteomics no Show all info Study aim Function/Drug delivery Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 95.4 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 38 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Size-exclusion chromatography Resin type Characterization: Protein analysis Protein Concentration Method Nanodrop Protein Yield (µg) number of particles per million cells Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD63/ CD81/ Flotillin1/ TSG101/ B-actin/ syntenin-1 Detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Particle yield number of particles per million cells

	EV210342	1/2	Homo sapiens	HEK293T	(d)(U)C	Tey SK	2022	56%
Study summary Full title ACE2-enriched extracellular vesicles enhance infectivity of live SARS-CoV-2 virus. All authors Tey SK, Lam H, Wong SWK, Zhao H, To KK, Yam JWP Journal J Extracell Vesicles Abstract NA (show more...)NA (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: TSG101/ CD63 non-EV: GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293T EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 70 Wash: time (min) 70 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins TSG101 Not detected contaminants GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 151 EV concentration Yes EM EM-type Immuno-EM EM protein CD63 Image type Close-up

	EV210342	2/2	Homo sapiens	HEK293T	(d)(U)C	Tey SK	2022	56%
Study summary Full title ACE2-enriched extracellular vesicles enhance infectivity of live SARS-CoV-2 virus. All authors Tey SK, Lam H, Wong SWK, Zhao H, To KK, Yam JWP Journal J Extracell Vesicles Abstract NA (show more...)NA (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin ACE2-overexpression Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: TSG101/ CD63 non-EV: GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293T EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 70 Wash: time (min) 70 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins TSG101 Not detected contaminants GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 154 EV concentration Yes EM EM-type Immuno-EM EM protein CD63 Image type Close-up

	EV210338	1/1	Bos taurus	1% skim milk	(d)(U)C	Fang, Zhou	2022	56%
Study summary Full title Small Extracellular Vesicles in Milk Cross the Blood-Brain Barrier in Murine Cerebral Cortex Endothelial Cells and Promote Dendritic Complexity in the Hippocampus and Brain Function in C57BL/6J Mice All authors Fang Zhou, Pearl Ebea, Ezra Mutai, Haichuan Wang, Sonal Sukreet, Shya Navazesh, Haluk Dogan, Wenhao Li, Juan Cui, Peng Ji, Denise M O Ramirez, Janos Zempleni Journal Frontiers Nutr. Abstract Human milk contains large amounts of small extracellular vesicles (sEVs) and their microRNA cargos, (show more...)Human milk contains large amounts of small extracellular vesicles (sEVs) and their microRNA cargos, whereas infant formulas contain only trace amounts of sEVs and microRNAs. We assessed the transport of sEVs across the blood-brain barrier (BBB) and sEV accumulation in distinct regions of the brain in brain endothelial cells and suckling mice. We further assessed sEV-dependent gene expression profiles and effects on the dendritic complexity of hippocampal granule cells and phenotypes of EV depletion in neonate, juvenile and adult mice. The transfer of sEVs across the BBB was assessed by using fluorophore-labeled bovine sEVs in brain endothelial bEnd.3 monolayers and dual chamber systems, and in wild-type newborn pups fostered to sEV and cargo tracking (ECT) dams that express sEVs labeled with a CD63-eGFP fusion protein for subsequent analysis by serial two-photon tomography and staining with anti-eGFP antibodies. Effects of EVs on gene expression and dendritic architecture of granule cells was analyzed in hippocampi from juvenile mice fed sEV and RNA-depleted (ERD) and sEV and RNA-sufficient (ERS) diets by using RNA-sequencing analysis and Golgi-Cox staining followed by integrated neuronal tracing and morphological analysis of neuronal dendrites, respectively. Spatial learning and severity of kainic acid-induced seizures were assessed in mice fed ERD and ERS diets. bEnd.3 cells internalized sEVs by using a saturable transport mechanism and secreted miR-34a across the basal membrane. sEVs penetrated the entire brain in fostering experiments; major regions of accumulation included the hippocampus, cortex and cerebellum. Two hundred ninety-five genes were differentially expressed in hippocampi from mice fed ERD and ERS diets; high-confidence gene networks included pathways implicated in axon guidance and calcium signaling. Juvenile pups fed the ERD diet had reduced dendritic complexity of dentate granule cells in the hippocampus, scored nine-fold lower in the Barnes maze test of spatial learning and memory, and the severity of seizures was 5-fold higher following kainic acid administration in adult mice fed the ERD diet compared to mice fed the ERS diet. We conclude that sEVs cross the BBB and contribute toward optimal neuronal development, spatial learning and memory, and resistance to kainic acid-induced seizures in mice. (hide) EV-METRIC 56% (16th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 1% skim milk Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD9/ CD63/ CD81/ Integrin-beta/ Histone H3/ Apolipoprotein B non-EV: None Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer Sample Species Bos taurus Sample Type 1% skim milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Fiberlite-F37L-8x100 rotor Pelleting: speed (g) 120,000 Wash: volume per pellet (ml) 50 Wash: time (min) 90 Wash: Rotor Type Fiberlite-F37-8x-100 rotor Wash: speed (g) 120,000 Characterization: Protein analysis Protein Concentration Method BCA/ Qubit Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ CD63/ CD81 Not detected EV-associated proteins Integrin-beta/ Histone H3/ Apolipoprotein B Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 106.6 +/- 6.7 EV concentration Yes EM EM-type Scanning-EM Image type Wide-field

	EV210237	2/2	Mus musculus	BV2	(d)(U)C	Gelibter S	2022	56%
Study summary Full title The impact of storage on extracellular vesicles: A systematic study. All authors Gelibter S, Marostica G, Mandelli A, Siciliani S, Podini P, Finardi A, Furlan R Journal J Extracell Vesicles Abstract Mounting evidence suggests that storage has an impact on extracellular vesicles (EVs) properties. Wh (show more...)Mounting evidence suggests that storage has an impact on extracellular vesicles (EVs) properties. While -80°C storage is a widespread approach, some authors proposed improved storage strategies with conflicting results. Here, we designed a systematic study to assess the impact of -80°C storage and freeze-thaw cycles on EVs. We tested the differences among eight storage strategies and investigated the possible fusion phenomena occurring during storage. EVs were collected from human plasma and murine microglia culture by size exclusion chromatography and ultracentrifugation, respectively. The analysis included: concentration, size and zeta potential (tunable resistive pulse sensing), contaminant protein assessment/ flow cytometry for the analysis of two single fluorescent-tagged EVs populations (GFP and mCherry), mixed before preservation. We found that -80°C storage reduces EVs concentration and sample purity in a time-dependent manner. Furthermore, it increases the particle size and size variability and modifies EVs zeta potential, with a shift of EVs in size-charge plots. None of the tested conditions prevented the observed effects. Freeze-thaw cycles lead to an EVs reduction after the first cycle and to a cycle-dependent increase in particle size. With flow cytometry, after storage, we observed a significant population of double-positive EVs (GFP -mCherry ). This observation may suggest the occurrence of fusion phenomena during storage. Our findings show a significant impact of storage on EVs samples in terms of particle loss, purity reduction and fusion phenomena leading to artefactual particles. Depending on downstream analyses and experimental settings, EVs should probably be processed from fresh, non-archival, samples in majority of cases. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NA Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Alix/ Flotillin-1/ Lamp1/ ANXA1/ IB4 non-EV: GM130/ H3 Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells BV2 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell count 40000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 4h Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 12 Wash: time (min) 960 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ Flotillin-1/ Lamp1/ ANXA1 Detected contaminants H3 Not detected contaminants GM130 Flow cytometry Type of Flow cytometry CytoflexS Calibration bead size 0.1, 0.3, 0.5, 0.7, 0.9 Antibody details provided? Yes Detected EV-associated proteins IB4 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 50-350 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.00e+11 Particle analysis: flow cytometry Flow cytometer type CytoflexS Hardware adjustment Calibration bead size 0.1, 0.3, 0.5, 0.7, 0.9 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.00e+7

	EV210145	1/1	Homo sapiens	human milk	(d)(U)C	Wang, John	2022	56%
Study summary Full title Quantitation of Exosomes and Their MicroRNA Cargos in Frozen Human Milk All authors Haichuan Wang, Di Wu, Sonal Sukreet, Anthony Delaney, Mandy Belfort, Janos Zempleni Journal Journal of pediatric gastroenterology and nutrition Abstract We assessed feasibility of analyzing exosomes and microRNA cargos in frozen human milk as a prerequi (show more...)We assessed feasibility of analyzing exosomes and microRNA cargos in frozen human milk as a prerequisite for epidemiological studies of milk exosomes. We collected milk from 5 mother-preterm infant dyads at 3 time points during postnatal hospital care for storage at −80 °C. We purified exosomes by ultracentrifugation, probed marker proteins using immunoblots, assessed size and counts with a nanoparticle tracker, and quantified 3 microRNAs with quantitative PCR. Positive exosome marker proteins were detectable; β-casein was the only detectable contaminant. Exosome count and size trended to decrease from early to late samples (count, 2.3 × 109 ± 3.8 × 109 to 5.6 × 108 ± 9.7 × 108 exosomes/mL; size, 117 ± 25 to 92 ± 16 nm). Two microRNAs were detectable in early samples only; cycle threshold values equaled 28.7 ± 0.7 for miR-30d-5p and miR-125a-5p; miR-423-5p was not detectable. We conclude that the analysis of exosomes and quantification of microRNAs is feasible in human milk previously stored at −80 °C. (hide) EV-METRIC 56% (62nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type human milk Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Alix/ CD63/ CD9 non-EV: beta casein/ alfa tubulin/ intergrin beta Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type human milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Thermo F37L-8x100 Pelleting: speed (g) 130000 Characterization: Protein analysis Protein Concentration Method Other Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ Alix Not detected contaminants tubulin-alfa/ integrin-beta/ casein-beta Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 117 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2000000000

	EV210024	10/12	Homo sapiens	Differentiated Glioblastoma Stem-like cells (DGC)	(d)(U)C	André-Grégoire, Gwennan	2022	56%
Study summary Full title Inhibition of the pseudokinase MLKL alters extracellular vesicle release and reduces tumor growth in glioblastoma All authors Gwennan André-Grégoire, Clément Maghe, Tiphaine Douanne, Sara, Rosińska, Fiorella Spinelli, An Thys, Kilian Trillet, Kathryn A.Jacobs, Cyndie Ballu, Aurélien Dupont, Anne-Marie Lyne, Florence M.G.Cavalli, Ignacio Busnelli, Vincent Hyenne, Jacky G.Goetz, Nicolas Bidère, Julie Gavard Journal iScience Abstract Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material fro (show more...)Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material from donor to recipient cells. EVs play key roles in glioblastoma progression because glioblastoma stem-like cells (GSCs) release pro-oncogenic, pro-angiogenic, and pro-inflammatory EVs. However, the molecular basis of EV release remains poorly understood. Here, we report the identification of the pseudokinase MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of EVs in GSCs. We find that genetic, protein, and pharmacological targeting of MLKL alters intracellular trafficking and EV release, and reduces GSC expansion. Nevertheless, this function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL reduces the tumor burden and the level of plasmatic EVs. This work highlights the necroptosis-independent role of MLKL in vesicle release and suggests that interfering with EVs is a promising therapeutic option to sensitize glioblastoma cells. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NSA Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD63 non-EV: GM130 Proteomics yes Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Differentiated Glioblastoma Stem-like cells (DGC) EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 11 Wash: time (min) 120 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD63 Not detected contaminants GM130 ELISA Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 125 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV190065	1/6	Homo sapiens	SW620	Filtration dUC	Wenzhe, Li	2022	56%
Study summary Full title Machine Learning-Assisted Dual-Marker Detection in Serum Small Extracellular Vesicles for the Diagnosis and Prognosis Prediction of Non-Small Cell Lung Cancer All authors Wenzhe Li, Ling Zhu, Kaidi Li, Siyuan Ye, Huayi Wang, Yadong Wang, Jianchao Xue, Chen Wang, Shanqing Li, Naixin Liang, Yanlian Yang Journal Nanomaterials Abstract Small extracellular vesicles (sEVs) carry molecular information from their source cells and are desi (show more...)Small extracellular vesicles (sEVs) carry molecular information from their source cells and are desired biomarkers for cancer diagnosis. We establish a machine learning-assisted dual-marker detection method to analyze the expression of epidermal growth factor receptor (EGFR) and C-X-C chemokine receptor 4 (CXCR4) in serum sEVs for the diagnosis and prognosis prediction of non-small cell lung cancer (NSCLC). We find that the serum sEV EGFR and CXCR4 are significantly higher in advanced stage NSCLC (A/NSCLC) patients compared to early stage NSCLC (E/NSCLC) patients and the healthy donors (HDs). A receiver operating characteristic curve (ROC) analysis demonstrates that the combination of EGFR and CXCR4 in serum sEVs as an efficient diagnostic index and malignant degree indicator for NSCLC. Machine learning further shows a diagnostic accuracy of 97.4% for the training cohort and 91.7% for the validation cohort based on the combinational marker. Moreover, this machine leaning-assisted serum sEV analysis successfully predicts the possibility of tumor relapse in three NSCLC patients by comparing their serum sEVs before and three days after surgery. This study provides an intelligent serum sEV-based assay for the diagnosis and prognosis prediction of NSCLC, and will benefit the precision management of NSCLC. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Filtration dUC Protein markers EV: EGFR/ CD81/ Flotillin-1 non-EV: Calnexin Proteomics no Show all info Study aim New methodological development/Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SW620 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 600 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 150000 Wash: volume per pellet (ml) 26,3 Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 150000 Filtration steps 0.22µm or 0.2µm Other Name other separation method dUC Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Detected EV-associated proteins EGFR/ CD81/ Flotillin-1 Not detected contaminants Calnexin Flow cytometry aspecific beads Antibody details provided? Yes Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins EGFR Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Not Reported NTA Report type Size range/distribution Reported size (nm) 121.2±65.9 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190065	2/6	Homo sapiens	A549	Filtration dUC	Wenzhe, Li	2022	56%
Study summary Full title Machine Learning-Assisted Dual-Marker Detection in Serum Small Extracellular Vesicles for the Diagnosis and Prognosis Prediction of Non-Small Cell Lung Cancer All authors Wenzhe Li, Ling Zhu, Kaidi Li, Siyuan Ye, Huayi Wang, Yadong Wang, Jianchao Xue, Chen Wang, Shanqing Li, Naixin Liang, Yanlian Yang Journal Nanomaterials Abstract Small extracellular vesicles (sEVs) carry molecular information from their source cells and are desi (show more...)Small extracellular vesicles (sEVs) carry molecular information from their source cells and are desired biomarkers for cancer diagnosis. We establish a machine learning-assisted dual-marker detection method to analyze the expression of epidermal growth factor receptor (EGFR) and C-X-C chemokine receptor 4 (CXCR4) in serum sEVs for the diagnosis and prognosis prediction of non-small cell lung cancer (NSCLC). We find that the serum sEV EGFR and CXCR4 are significantly higher in advanced stage NSCLC (A/NSCLC) patients compared to early stage NSCLC (E/NSCLC) patients and the healthy donors (HDs). A receiver operating characteristic curve (ROC) analysis demonstrates that the combination of EGFR and CXCR4 in serum sEVs as an efficient diagnostic index and malignant degree indicator for NSCLC. Machine learning further shows a diagnostic accuracy of 97.4% for the training cohort and 91.7% for the validation cohort based on the combinational marker. Moreover, this machine leaning-assisted serum sEV analysis successfully predicts the possibility of tumor relapse in three NSCLC patients by comparing their serum sEVs before and three days after surgery. This study provides an intelligent serum sEV-based assay for the diagnosis and prognosis prediction of NSCLC, and will benefit the precision management of NSCLC. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Filtration dUC Protein markers EV: EGFR/ CD81/ Flotillin-1/ CXCR4 non-EV: Calnexin Proteomics no Show all info Study aim New methodological development/Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells A549 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 600 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 150000 Wash: volume per pellet (ml) 26,3 Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 150000 Filtration steps 0.22µm or 0.2µm Other Name other separation method dUC Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Detected EV-associated proteins EGFR/ CD81/ Flotillin-1/ CXCR4 Not detected contaminants Calnexin Flow cytometry aspecific beads Antibody details provided? Yes Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins EGFR/ CXCR4 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Not Reported NTA Report type Size range/distribution Reported size (nm) 121.2±65.9 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190065	3/6	Homo sapiens	H1975	Filtration dUC	Wenzhe, Li	2022	56%
Study summary Full title Machine Learning-Assisted Dual-Marker Detection in Serum Small Extracellular Vesicles for the Diagnosis and Prognosis Prediction of Non-Small Cell Lung Cancer All authors Wenzhe Li, Ling Zhu, Kaidi Li, Siyuan Ye, Huayi Wang, Yadong Wang, Jianchao Xue, Chen Wang, Shanqing Li, Naixin Liang, Yanlian Yang Journal Nanomaterials Abstract Small extracellular vesicles (sEVs) carry molecular information from their source cells and are desi (show more...)Small extracellular vesicles (sEVs) carry molecular information from their source cells and are desired biomarkers for cancer diagnosis. We establish a machine learning-assisted dual-marker detection method to analyze the expression of epidermal growth factor receptor (EGFR) and C-X-C chemokine receptor 4 (CXCR4) in serum sEVs for the diagnosis and prognosis prediction of non-small cell lung cancer (NSCLC). We find that the serum sEV EGFR and CXCR4 are significantly higher in advanced stage NSCLC (A/NSCLC) patients compared to early stage NSCLC (E/NSCLC) patients and the healthy donors (HDs). A receiver operating characteristic curve (ROC) analysis demonstrates that the combination of EGFR and CXCR4 in serum sEVs as an efficient diagnostic index and malignant degree indicator for NSCLC. Machine learning further shows a diagnostic accuracy of 97.4% for the training cohort and 91.7% for the validation cohort based on the combinational marker. Moreover, this machine leaning-assisted serum sEV analysis successfully predicts the possibility of tumor relapse in three NSCLC patients by comparing their serum sEVs before and three days after surgery. This study provides an intelligent serum sEV-based assay for the diagnosis and prognosis prediction of NSCLC, and will benefit the precision management of NSCLC. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Filtration dUC Protein markers EV: EGFR/ CD81/ Flotillin-1/ CXCR4 non-EV: Calnexin Proteomics no Show all info Study aim New methodological development/Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells H1975 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 600 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 150000 Wash: volume per pellet (ml) 26,3 Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 150000 Filtration steps 0.22µm or 0.2µm Other Name other separation method dUC Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Detected EV-associated proteins EGFR/ CD81/ Flotillin-1/ CXCR4 Not detected contaminants Calnexin Flow cytometry aspecific beads Antibody details provided? Yes Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins EGFR/ CXCR4 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Not Reported NTA Report type Size range/distribution Reported size (nm) 131.1±53.9 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: 1.00e+0 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190065	4/6	Homo sapiens	H1650	Filtration dUC	Wenzhe, Li	2022	56%
Study summary Full title Machine Learning-Assisted Dual-Marker Detection in Serum Small Extracellular Vesicles for the Diagnosis and Prognosis Prediction of Non-Small Cell Lung Cancer All authors Wenzhe Li, Ling Zhu, Kaidi Li, Siyuan Ye, Huayi Wang, Yadong Wang, Jianchao Xue, Chen Wang, Shanqing Li, Naixin Liang, Yanlian Yang Journal Nanomaterials Abstract Small extracellular vesicles (sEVs) carry molecular information from their source cells and are desi (show more...)Small extracellular vesicles (sEVs) carry molecular information from their source cells and are desired biomarkers for cancer diagnosis. We establish a machine learning-assisted dual-marker detection method to analyze the expression of epidermal growth factor receptor (EGFR) and C-X-C chemokine receptor 4 (CXCR4) in serum sEVs for the diagnosis and prognosis prediction of non-small cell lung cancer (NSCLC). We find that the serum sEV EGFR and CXCR4 are significantly higher in advanced stage NSCLC (A/NSCLC) patients compared to early stage NSCLC (E/NSCLC) patients and the healthy donors (HDs). A receiver operating characteristic curve (ROC) analysis demonstrates that the combination of EGFR and CXCR4 in serum sEVs as an efficient diagnostic index and malignant degree indicator for NSCLC. Machine learning further shows a diagnostic accuracy of 97.4% for the training cohort and 91.7% for the validation cohort based on the combinational marker. Moreover, this machine leaning-assisted serum sEV analysis successfully predicts the possibility of tumor relapse in three NSCLC patients by comparing their serum sEVs before and three days after surgery. This study provides an intelligent serum sEV-based assay for the diagnosis and prognosis prediction of NSCLC, and will benefit the precision management of NSCLC. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Filtration dUC Protein markers EV: CD81/ Flotillin-1/ CXCR4 non-EV: Calnexin Proteomics no Show all info Study aim New methodological development/Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells H1650 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 600 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 150000 Wash: volume per pellet (ml) 26,3 Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 150000 Filtration steps 0.22µm or 0.2µm Other Name other separation method dUC Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Detected EV-associated proteins CD81/ Flotillin-1/ CXCR4 Not detected contaminants Calnexin Flow cytometry aspecific beads Antibody details provided? Yes Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins CXCR4 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Not Reported NTA Report type Size range/distribution Reported size (nm) 131.1±53.9 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: 1.00e+0 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190047	1/10	Homo sapiens	MDA-MB-231	(d)(U)C ExoQuick UF	Xu, Jing	2022	56%
Study summary Full title Chloroquine treatment induces secretion of autophagy-related proteins and inclusion of Atg8-family proteins in distinct extracellular vesicle populations All authors Jing Xu, Kevin C Yang, Nancy Erro Go, Shane Colborne, Cally J Ho, Elham Hosseini-Beheshti, Alf H Lystad, Anne Simonsen, Emma Tomlinson Guns, Gregg B Morin, Sharon M Gorski Journal Autophagy Abstract Chloroquine (CQ), a lysosomotropic agent, is commonly used to inhibit lysosomal degradation and macr (show more...)Chloroquine (CQ), a lysosomotropic agent, is commonly used to inhibit lysosomal degradation and macroautophagy/autophagy. Here we investigated the cell-extrinsic effects of CQ on secretion. We showed that lysosomal and autophagy inhibition by CQ altered the secretome, and induced the release of Atg8 orthologs and autophagy receptors. Atg8-family proteins, in particular, were secreted inside small extracellular vesicles (sEVs) in a lipidation-dependent manner. CQ treatment enhanced the release of Atg8-family proteins inside sEVs. Using full-length ATG16L1 and an ATG16L1 mutant that enables Atg8-family protein lipidation on double but not on single membranes, we demonstrated that LC3B is released in two distinct sEV populations: one enriched with SDCBP/Syntenin-1, CD63, and endosomal lipidated LC3B, and another that contains LC3B but is not enriched with SDCBP/Syntenin-1 or CD63, and which our data supports as originating from a double-membrane source. Our findings underscore the context-dependency of sEV heterogeneity and composition, and illustrate the integration of autophagy and sEV composition in response to lysosomal inhibition.Abbreviations: ACTB: actin beta; ANOVA: analysis of variance; ATG4B: autophagy related 4B cysteine peptidase; Atg8: autophagy related 8; ATG16L1: autophagy related 16 like 1; ATP5F1A/ATP5a: ATP synthase F1 subunit alpha; CALCOCO2: calcium binding and coiled-coil domain 2; CASP3: caspase 3; CASP7: caspase 7; CQ: chloroquine; CD9: CD9 molecule; CD63: CD63 molecule; DAPI: 4',6-diamidino-2-phenylindole; DQ-BSA: dye quenched-bovine serum albumin; ER: endoplasmic reticulum; ERN1/IRE1a: endoplasmic reticulum to nucleus signaling 1; EV: extracellular vesicles; FBS: fetal bovine serum; FDR: false discovery rate; GABARAP: GABA type A receptor-associated protein; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GO: gene ontology; HCQ: hydroxychloroquine; HSP90AA1: heat shock protein 90 alpha family class A member 1; IP: immunoprecipitation; KO: knockout; LAMP2: lysosomal associated membrane protein 2; LIR: LC3-interacting region; LMNA: lamin A/C; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MS: mass spectrometry; NBR1: NBR1 autophagy cargo receptor; NCOA4: nuclear receptor coactivator 4; NTA: nanoparticle tracking analysis; PE: phosphatidylethanolamine; PECA: probe-level expression change averaging; SDCBP/syntenin-1: syndecan binding protein; SD: standard deviation; SE: secreted; sEV: small extracellular vesicles; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TEM: transmission electron microscopy; TMT: tandem-mass tag; TSG101: tumor susceptibility 101; ULK1: unc-51 like autophagy activating kinase 1; WC: whole cell. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C ExoQuick UF Protein markers EV: TSG101/ CD63/ HSP90/ LAMP2/ CD9/ Syntenin-1 non-EV: IRE1a Proteomics yes Show all info Study aim Function/Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDA-MB-231 EV-harvesting Medium Serum free medium Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ Syntenin-1/ LAMP2/ HSP90/ TSG101 Not detected contaminants IRE1a Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 134.1 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV190047	4/10	Homo sapiens	MDA-MB-231	(d)(U)C ExoQuick UF	Xu, Jing	2022	56%
Study summary Full title Chloroquine treatment induces secretion of autophagy-related proteins and inclusion of Atg8-family proteins in distinct extracellular vesicle populations All authors Jing Xu, Kevin C Yang, Nancy Erro Go, Shane Colborne, Cally J Ho, Elham Hosseini-Beheshti, Alf H Lystad, Anne Simonsen, Emma Tomlinson Guns, Gregg B Morin, Sharon M Gorski Journal Autophagy Abstract Chloroquine (CQ), a lysosomotropic agent, is commonly used to inhibit lysosomal degradation and macr (show more...)Chloroquine (CQ), a lysosomotropic agent, is commonly used to inhibit lysosomal degradation and macroautophagy/autophagy. Here we investigated the cell-extrinsic effects of CQ on secretion. We showed that lysosomal and autophagy inhibition by CQ altered the secretome, and induced the release of Atg8 orthologs and autophagy receptors. Atg8-family proteins, in particular, were secreted inside small extracellular vesicles (sEVs) in a lipidation-dependent manner. CQ treatment enhanced the release of Atg8-family proteins inside sEVs. Using full-length ATG16L1 and an ATG16L1 mutant that enables Atg8-family protein lipidation on double but not on single membranes, we demonstrated that LC3B is released in two distinct sEV populations: one enriched with SDCBP/Syntenin-1, CD63, and endosomal lipidated LC3B, and another that contains LC3B but is not enriched with SDCBP/Syntenin-1 or CD63, and which our data supports as originating from a double-membrane source. Our findings underscore the context-dependency of sEV heterogeneity and composition, and illustrate the integration of autophagy and sEV composition in response to lysosomal inhibition.Abbreviations: ACTB: actin beta; ANOVA: analysis of variance; ATG4B: autophagy related 4B cysteine peptidase; Atg8: autophagy related 8; ATG16L1: autophagy related 16 like 1; ATP5F1A/ATP5a: ATP synthase F1 subunit alpha; CALCOCO2: calcium binding and coiled-coil domain 2; CASP3: caspase 3; CASP7: caspase 7; CQ: chloroquine; CD9: CD9 molecule; CD63: CD63 molecule; DAPI: 4',6-diamidino-2-phenylindole; DQ-BSA: dye quenched-bovine serum albumin; ER: endoplasmic reticulum; ERN1/IRE1a: endoplasmic reticulum to nucleus signaling 1; EV: extracellular vesicles; FBS: fetal bovine serum; FDR: false discovery rate; GABARAP: GABA type A receptor-associated protein; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GO: gene ontology; HCQ: hydroxychloroquine; HSP90AA1: heat shock protein 90 alpha family class A member 1; IP: immunoprecipitation; KO: knockout; LAMP2: lysosomal associated membrane protein 2; LIR: LC3-interacting region; LMNA: lamin A/C; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MS: mass spectrometry; NBR1: NBR1 autophagy cargo receptor; NCOA4: nuclear receptor coactivator 4; NTA: nanoparticle tracking analysis; PE: phosphatidylethanolamine; PECA: probe-level expression change averaging; SDCBP/syntenin-1: syndecan binding protein; SD: standard deviation; SE: secreted; sEV: small extracellular vesicles; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TEM: transmission electron microscopy; TMT: tandem-mass tag; TSG101: tumor susceptibility 101; ULK1: unc-51 like autophagy activating kinase 1; WC: whole cell. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 10uM CQ Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C ExoQuick UF Protein markers EV: TSG101/ CD63/ HSP90/ LAMP2/ CD9/ Syntenin-1 non-EV: IRE1a Proteomics yes Show all info Study aim Function/Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDA-MB-231 EV-harvesting Medium Serum free medium Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins Syntenin-1/ LAMP2/ CD9/ CD63/ HSP90/ TSG101 Not detected contaminants IRE1a Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 130.7 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV210301	1/1	Homo sapiens	Expi293F	(d)(U)C DG	McConnell, Russell	2022	55%
Study summary Full title Transfection reagent artefact likely accounts for some reports of extracellular vesicle function All authors Russell E. McConnell, Madeleine Youniss, Bhargavee Gnanasambandam, Palak Shah, Wei Zhang, Jonathan D. Finn Journal J Extracell Vesicles Abstract Extracellular vesicles (EV) are important mediators of cell communication and physiology. EVs are fr (show more...)Extracellular vesicles (EV) are important mediators of cell communication and physiology. EVs are frequently investigated by transiently transfecting cells with plasmid DNA to produce EVs modified with protein(s) or nucleic acid(s) of interest. DNA-transfection reagent complexes (DTC) are approximately the same size as EVs, raising the possibility that some purification procedures may fail to separate these two species and activity arising from carryover DTC may be improperly attributed to EVs. We find that differential ultracentrifugation, a commonly employed EV isolation procedure, does not separate EVs from DTC present in the cell culture supernatant of transiently transfected cells. We demonstrate that the biological activity of an EV-directed Cre recombinase is due to contaminating plasmid DNA and not EV-mediated delivery of Cre protein. Moreover, steps commonly taken to remove plasmid DNA from EV samples, such as media exchanges and treatment with nucleases, are ineffective at avoiding this artefact. Due to the pernicious nature of plasmid DNA in these cellular assays, some reports of EV function are likely artefacts produced by contaminating DTC. EVs and DTC can be separated by density gradient ultracentrifugation, highlighting the importance of validating elimination of DTC when using transient transfection of EV-producing cells to interrogate EV function. (hide) EV-METRIC 55% (88th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: CD9/ CD63/ CD81 non-EV: None Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 90 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 133,9 Wash: volume per pellet (ml) 13 Wash: time (min) 180 Wash: Rotor Type SW 41 Ti Wash: speed (g) 150 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 18 Highest density fraction 45 Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 150000 Duration (min) 960 Fraction processing Centrifugation Pelleting: volume per fraction 13 Pelleting: duration (min) 120 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 133900 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 210 EV concentration Yes

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