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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220405	1/3	Homo sapiens	Cerebrospinal Fluid	UF qEV	Ursula S Sandau	2022	75%
Study summary Full title Differential Effects of APOE Genotype on MicroRNA Cargo of Cerebrospinal Fluid Extracellular Vesicles in Females With Alzheimer's Disease Compared to Males All authors Ursula S Sandau, Trevor J McFarland, Sierra J Smith, Douglas R Galasko, Joseph F Quinn, Julie A Saugstad Journal Abstract Multiple biological factors, including age, sex, and genetics, influence Alzheimer's disease (AD) ri (show more...)Multiple biological factors, including age, sex, and genetics, influence Alzheimer's disease (AD) risk. Of the 6.2 million Americans living with Alzheimer's dementia in 2021, 3.8 million are women and 2.4 million are men. The strongest genetic risk factor for sporadic AD is apolipoprotein E-e4 (APOE-e4). Female APOE-e4 carriers develop AD more frequently than age-matched males and have more brain atrophy and memory loss. Consequently, biomarkers that are sensitive to biological risk factors may improve AD diagnostics and may provide insight into underlying mechanistic changes that could drive disease progression. Here, we have assessed the effects of sex and APOE-e4 on the miRNA cargo of cerebrospinal fluid (CSF) extracellular vesicles (EVs) in AD. We used ultrafiltration (UF) combined with size exclusion chromatography (SEC) to enrich CSF EVs (e.g., Flotillin+). CSF EVs were isolated from female and male AD or controls (CTLs) that were either APOE-e3,4 or -e3,3 positive (n = 7/group, 56 total). MiRNA expression levels were quantified using a custom TaqMan™ array that assayed 190 miRNAs previously found in CSF, including 25 miRNAs that we previously validated as candidate AD biomarkers. We identified changes in the EV miRNA cargo that were affected by both AD and sex. In total, four miRNAs (miR-16-5p, -331-3p, -409-3p, and -454-3p) were significantly increased in AD vs. CTL, independent of sex and APOE-e4 status. Pathway analysis of the predicted gene targets of these four miRNAs with identified pathways was highly relevant to neurodegeneration (e.g., senescence and autophagy). There were also three miRNAs (miR-146b-5p, -150-5p, and -342-3p) that were significantly increased in females vs. males, independent of disease state and APOE-e4 status. We then performed a statistical analysis to assess the effect of APOE genotype in AD within each sex and found that APOE-e4 status affects different subsets of CSF EV miRNAs in females vs. males. Together, this study demonstrates the complexity of the biological factors associated with AD risk and the impact on EV miRNAs, which may contribute to AD pathophysiology. (hide) EV-METRIC 75% (98th 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 Cerebrospinal Fluid 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 qEV Protein markers EV: CD9/ CD63/ CD81/ Flotillin1/ TSG101/ Annexin V/ Glast/ CD11b/ NCAM-1/ Synaptophysin/ TMEM119 non-EV: Albumin/ APOA1/ APOE Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cerebrospinal Fluid Separation Method Ultra filtration Cut-off size (kDa) 30 Membrane type Regenerated cellulose Commercial kit qEV (35 nm) Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Fluorometric assay Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81/ Flotillin1/ TSG101/ Annexin V/ Glast/ CD11b Not detected EV-associated proteins NCAM-1/ Synaptophysin/ TMEM119 Detected contaminants APOE Not detected contaminants Albumin/ APOA1 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 76-620 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.50E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-100

	EV220163	1/3	Homo sapiens	Serum	qEV70	Lauren A. Newman	2022	75%
Study summary Full title Addressing MISEV guidance using targeted LC-MS/MS: A method for the detection and quantification of extracellular vesicle-enriched and contaminant protein markers from blood All authors Lauren A. Newman, Zivile Useckaite, Andrew Rowland Journal Journal of Extracellular Biology Abstract Extracellular vesicles (EVs) are membrane-bound nanosized particles released by cells into bodily fl (show more...)Extracellular vesicles (EVs) are membrane-bound nanosized particles released by cells into bodily fluids containing an array of molecular cargo. Several characteristics, including stability and accessibility in biofluids such as blood and urine, make EVs and associated cargo attractive biomarkers and therapeutic tools. To promote robust characterisation of EV isolates, the minimal requirements for the study of extracellular vesicles (MISEV) guidelines recommend the analysis of proteins in EV samples, including positive EV-associated markers and negative contaminant markers based on commonly co-isolated components of the starting material. Western blot is conventionally used to address the guidelines; however, this approach is limited in terms of quantitation and throughput and requires larger volumes than typically available for patient samples. The increasing application of EVs as liquid biopsy in clinical contexts requires a high-throughput multiplexed approach for analysis of protein markers from small volumes of starting material. Here, we document the development and validation of a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for the quantification of markers associated with EVs and non-vesicle contaminants from human blood samples. The assay was highly sensitive, requiring only a fraction of the sample consumed for immunoblots, fully quantitative and high throughput. Application of the assay to EVs isolated by size exclusion chromatography (SEC) and precipitation revealed differences in yield, purity and recovery of subpopulations. (hide) EV-METRIC 75% (98th 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 qEV70 Protein markers EV: CD81/ TSG101/ CD9 non-EV: Calnexin/ Albumin Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Serum Separation Method Commercial kit qEV70 Other Name other separation method qEV70 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 CD81/ TSG101 Not detected EV-associated proteins CD9 Detected contaminants Albumin Not detected contaminants Calnexin Other 2 Targeted LC-MS Detected EV-associated proteins CD9/ CD81/ TSG101 Detected contaminants Albumin/ Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 101.7 EV concentration Yes Particle yield particles per milliliter of starting sample: 8.33E11 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 70

	EV220163	2/3	Homo sapiens	Serum	ExoQuick	Lauren A. Newman	2022	75%
Study summary Full title Addressing MISEV guidance using targeted LC-MS/MS: A method for the detection and quantification of extracellular vesicle-enriched and contaminant protein markers from blood All authors Lauren A. Newman, Zivile Useckaite, Andrew Rowland Journal Journal of Extracellular Biology Abstract Extracellular vesicles (EVs) are membrane-bound nanosized particles released by cells into bodily fl (show more...)Extracellular vesicles (EVs) are membrane-bound nanosized particles released by cells into bodily fluids containing an array of molecular cargo. Several characteristics, including stability and accessibility in biofluids such as blood and urine, make EVs and associated cargo attractive biomarkers and therapeutic tools. To promote robust characterisation of EV isolates, the minimal requirements for the study of extracellular vesicles (MISEV) guidelines recommend the analysis of proteins in EV samples, including positive EV-associated markers and negative contaminant markers based on commonly co-isolated components of the starting material. Western blot is conventionally used to address the guidelines; however, this approach is limited in terms of quantitation and throughput and requires larger volumes than typically available for patient samples. The increasing application of EVs as liquid biopsy in clinical contexts requires a high-throughput multiplexed approach for analysis of protein markers from small volumes of starting material. Here, we document the development and validation of a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for the quantification of markers associated with EVs and non-vesicle contaminants from human blood samples. The assay was highly sensitive, requiring only a fraction of the sample consumed for immunoblots, fully quantitative and high throughput. Application of the assay to EVs isolated by size exclusion chromatography (SEC) and precipitation revealed differences in yield, purity and recovery of subpopulations. (hide) EV-METRIC 75% (98th 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 ExoQuick Protein markers EV: CD81/ TSG101/ CD9 non-EV: Calnexin/ Albumin Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Serum Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method BCA 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 CD81/ TSG101 Not detected EV-associated proteins CD9 Detected contaminants Albumin Not detected contaminants Calnexin Other 2 Targeted LC-MS Detected EV-associated proteins CD81/ TSG101 Not detected EV-associated proteins CD9 Detected contaminants Albumin Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 111.7 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.31E13 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 150

	EV220163	3/3	Homo sapiens	Serum	qEV35	Lauren A. Newman	2022	75%
Study summary Full title Addressing MISEV guidance using targeted LC-MS/MS: A method for the detection and quantification of extracellular vesicle-enriched and contaminant protein markers from blood All authors Lauren A. Newman, Zivile Useckaite, Andrew Rowland Journal Journal of Extracellular Biology Abstract Extracellular vesicles (EVs) are membrane-bound nanosized particles released by cells into bodily fl (show more...)Extracellular vesicles (EVs) are membrane-bound nanosized particles released by cells into bodily fluids containing an array of molecular cargo. Several characteristics, including stability and accessibility in biofluids such as blood and urine, make EVs and associated cargo attractive biomarkers and therapeutic tools. To promote robust characterisation of EV isolates, the minimal requirements for the study of extracellular vesicles (MISEV) guidelines recommend the analysis of proteins in EV samples, including positive EV-associated markers and negative contaminant markers based on commonly co-isolated components of the starting material. Western blot is conventionally used to address the guidelines; however, this approach is limited in terms of quantitation and throughput and requires larger volumes than typically available for patient samples. The increasing application of EVs as liquid biopsy in clinical contexts requires a high-throughput multiplexed approach for analysis of protein markers from small volumes of starting material. Here, we document the development and validation of a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for the quantification of markers associated with EVs and non-vesicle contaminants from human blood samples. The assay was highly sensitive, requiring only a fraction of the sample consumed for immunoblots, fully quantitative and high throughput. Application of the assay to EVs isolated by size exclusion chromatography (SEC) and precipitation revealed differences in yield, purity and recovery of subpopulations. (hide) EV-METRIC 75% (98th 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 qEV35 Protein markers EV: CD81/ TSG101/ CD9 non-EV: Calnexin/ Albumin Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Serum Separation Method Commercial kit qEV35 Other Name other separation method qEV35 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 CD81/ TSG101 Not detected EV-associated proteins CD9 Detected contaminants Albumin Not detected contaminants Calnexin Other 2 Targeted LC-MS Detected EV-associated proteins CD9/ CD81/ TSG101 Detected contaminants Albumin/ Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 95.53 EV concentration Yes Particle yield particles per milliliter of starting sample: 8.20E12 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 70

	EV220120	1/2	Homo sapiens	primary macrophages	UF qEV	Pantazi P	2022	75%
Study summary Full title Distinct non-coding RNA cargo of extracellular vesicles from M1 and M2 human primary macrophages. All authors Pantazi P, Clements T, Venø M, Abrahams VM, Holder B Journal J Extracell Vesicles Abstract Macrophages are important antigen presenting cells which can release extracellular vesicles (EVs) ca (show more...)Macrophages are important antigen presenting cells which can release extracellular vesicles (EVs) carrying functional cargo including non-coding RNAs. Macrophages can be broadly classified into M1 'classical' and M2 'alternatively-activated' macrophages. M1 macrophages have been linked with inflammation-associated pathologies, whereas a switch towards an M2 phenotype indicates resolution of inflammation and tissue regeneration. Here, we provide the first comprehensive analysis of the small RNA cargo of EVs from human M1 and M2 primary macrophages. Using small RNA sequencing, we identified several types of small non-coding RNAs in M1 and M2 macrophage EVs including miRNAs, isomiRs, tRNA fragments, piRNA, snRNA, snoRNA and Y-RNA fragments. Distinct differences were observed between M1 and M2 EVs, with higher relative abundance of miRNAs, and lower abundance of tRNA fragments in M1 compared to M2 EVs. MicroRNA-target enrichment analysis identified several gene targets involved in gene expression and inflammatory signalling pathways. EVs were also enriched in tRNA fragments, primarily originating from the 5' end or the internal region of the full length tRNAs, many of which were differentially abundant in M1 and M2 EVs. Similarly, several other small non-coding RNAs, namely snRNAs, snoRNAs and Y-RNA fragments, were differentially enriched in M1 and M2 EVs/ we discuss their putative roles in macrophage EVs. In conclusion, we show that M1 and M2 macrophages release EVs with distinct RNA cargo, which has the potential to contribute to the unique effect of these cell subsets on their microenvironment. (hide) EV-METRIC 75% (96th 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 M1 pathway 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 Protein markers EV: Alix/ MHC1/ MHC2/ Fibronectin/ HSP70/ CD63/ CD81/ Syntenin-1 non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary macrophages EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method Ultra filtration Cut-off size (kDa) 30 Commercial kit qEV 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/ MHC1/ MHC2/ Fibronectin Not detected EV-associated proteins HSP70 Not detected contaminants Calnexin ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ CD63/ CD81/ MHC1/ Syntenin-1 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ RNAsequencing Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 162.4 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.25E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220120	2/2	Homo sapiens	primary macrophages	UF qEV	Pantazi P	2022	75%
Study summary Full title Distinct non-coding RNA cargo of extracellular vesicles from M1 and M2 human primary macrophages. All authors Pantazi P, Clements T, Venø M, Abrahams VM, Holder B Journal J Extracell Vesicles Abstract Macrophages are important antigen presenting cells which can release extracellular vesicles (EVs) ca (show more...)Macrophages are important antigen presenting cells which can release extracellular vesicles (EVs) carrying functional cargo including non-coding RNAs. Macrophages can be broadly classified into M1 'classical' and M2 'alternatively-activated' macrophages. M1 macrophages have been linked with inflammation-associated pathologies, whereas a switch towards an M2 phenotype indicates resolution of inflammation and tissue regeneration. Here, we provide the first comprehensive analysis of the small RNA cargo of EVs from human M1 and M2 primary macrophages. Using small RNA sequencing, we identified several types of small non-coding RNAs in M1 and M2 macrophage EVs including miRNAs, isomiRs, tRNA fragments, piRNA, snRNA, snoRNA and Y-RNA fragments. Distinct differences were observed between M1 and M2 EVs, with higher relative abundance of miRNAs, and lower abundance of tRNA fragments in M1 compared to M2 EVs. MicroRNA-target enrichment analysis identified several gene targets involved in gene expression and inflammatory signalling pathways. EVs were also enriched in tRNA fragments, primarily originating from the 5' end or the internal region of the full length tRNAs, many of which were differentially abundant in M1 and M2 EVs. Similarly, several other small non-coding RNAs, namely snRNAs, snoRNAs and Y-RNA fragments, were differentially enriched in M1 and M2 EVs/ we discuss their putative roles in macrophage EVs. In conclusion, we show that M1 and M2 macrophages release EVs with distinct RNA cargo, which has the potential to contribute to the unique effect of these cell subsets on their microenvironment. (hide) EV-METRIC 75% (96th 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 M2 pathway 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 Protein markers EV: Alix/ MHC1/ MHC2/ Fibronectin/ HSP70/ CD63/ CD81/ Syntenin-1 non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary macrophages EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method Ultra filtration Cut-off size (kDa) 30 Commercial kit qEV 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/ MHC1/ MHC2/ Fibronectin Not detected EV-associated proteins HSP70 Not detected contaminants Calnexin ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ CD63/ CD81/ MHC1/ Syntenin-1 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ RNAsequencing Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 155.4 EV concentration Yes Particle yield particles per milliliter of starting sample: 6.57E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220003	1/2	Homo sapiens	Blood plasma	DG SEC (non-commercial)	Karimi, Nasibeh	2022	75%
Study summary Full title Tetraspanins distinguish separate extracellular vesicle subpopulations in human serum and plasma - Contributions of platelet extracellular vesicles in plasma samples All authors Nasibeh Karimi, Razieh Dalirfardouei, Tomás Dias, Jan Lötvall, Cecilia Lässer Journal J Extracell Vesicles Abstract Background: The ability to isolate extracellular vesicles (EVs) from blood is vital in the developme (show more...)Background: The ability to isolate extracellular vesicles (EVs) from blood is vital in the development of EVs as disease biomarkers. Both serum and plasma can be used, but few studies have compared these sources in terms of the type of EVs that are obtained. The aim of this study was to determine the presence of different subpopulations of EVs in plasma and serum. Method: Blood was collected from healthy subjects, and plasma and serum were isolated in parallel. ACD or EDTA tubes were used for the collection of plasma, while serum was obtained in clot activator tubes. EVs were isolated utilising a combination of density cushion and SEC, a combination of density cushion and gradient or by a bead antibody capturing system (anti-CD63, anti-CD9 and anti-CD81 beads). The subpopulations of EVs were analysed by NTA, Western blot, SP-IRIS, conventional and nano flow cytometry, magnetic bead ELISA and mass spectrometry. Additionally, different isolation protocols for plasma were compared to determine the contribution of residual platelets in the analysis. Results: This study shows that a higher number of CD9+ EVs were present in EDTA-plasma compared to ACD-plasma and to serum, and the presence of CD41a on these EVs suggests that they were released from platelets. Furthermore, only a very small number of EVs in blood were double-positive for CD63 and CD81. The CD63+ EVs were enriched in serum, while CD81+ vesicles were the rarest subpopulation in both plasma and serum. Additionally, EDTA-plasma contained more residual platelets than ACD-plasma and serum, and two centrifugation steps were crucial to reduce the number of platelets in plasma prior to EV isolation. Conclusion: These results show that human blood contains multiple subpopulations of EVs that carry different tetraspanins. Blood sampling methods, including the use of anti-coagulants and choice of centrifugation protocols, can affect EV analyses and should always be reported in detail. (hide) EV-METRIC 75% (96th 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 Blood plasma 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 Density gradient Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD63/ CD81/ Flotillin-1/ CD41a/ P-selectin non-EV: Calnexin Proteomics no EV density (g/ml) 1.06-1.16 Show all info Study aim Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 50% Total gradient volume, incl. sample (mL) 12 ml Sample volume (mL) 6 ml Orientation Top-down Rotor type SW 41 Ti Speed (g) 178,000 Duration (min) 180 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) 27.8 Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81/ Flotillin-1/ CD41a/ P-selectin Not detected contaminants Calnexin Detected EV-associated proteins CD9/ CD63/ CD81/ CD41a Detected EV-associated proteins CD9/ CD63/ CD81/ CD41a Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield as total number of particles in pooled SEC fractions from 6 mL of sample: 2.90e+9 Report type Size range/distribution Report size 50-70 EV-concentration No

	EV210383	1/2	Mus musculus	Serum	ExoQuick	Mkrtchian, Soiuren	2022	75%
Study summary Full title Surgical Trauma in Mice Modifies the Content of Circulating Extracellular Vesicles All authors Souren Mkrtchian, Anette Ebberyd, Rosanne E. Veerman, María Méndez-Lago, Susanne Gabrielsson, Lars I. Eriksson, and Marta Gómez-Galán Journal Front Immunol Abstract Surgical interventions rapidly trigger a cascade of molecular, cellular, and neural signaling respon (show more...)Surgical interventions rapidly trigger a cascade of molecular, cellular, and neural signaling responses that ultimately reach remote organs, including the brain. Using a mouse model of orthopedic surgery, we have previously demonstrated hippocampal metabolic, structural, and functional changes associated with cognitive impairment. However, the nature of the underlying signals responsible for such periphery-to-brain communication remains hitherto elusive. Here we present the first exploratory study that tests the hypothesis of extracellular vesicles (EVs) as potential mediators carrying information from the injured tissue to the distal organs including the brain. The primary goal was to investigate whether the cargo of circulating EVs after surgery can undergo quantitative changes that could potentially trigger phenotypic modifications in the target tissues. EVs were isolated from the serum of the mice subjected to a tibia surgery after 6, 24, and 72 h, and the proteome and miRNAome were investigated using mass spectrometry and RNA-seq approaches. We found substantial differential expression of proteins and miRNAs starting at 6 h post-surgery and peaking at 24 h. Interestingly, one of the up-regulated proteins at 24 h was α-synuclein, a pathogenic hallmark of certain neurodegenerative syndromes. Analysis of miRNA target mRNA and corresponding biological pathways indicate the potential of post-surgery EVs to modify the extracellular matrix of the recipient cells and regulate metabolic processes including fatty acid metabolism. We conclude that surgery alters the cargo of circulating EVs in the blood, and our results suggest EVs as potential systemic signal carriers mediating remote effects of surgery on the brain. (hide) EV-METRIC 75% (98th 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 ExoQuick Protein markers EV: CD63/ CD81/ HSP70 non-EV: Albumin/ Argonaute?2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Mus musculus Sample Type Serum Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) 0.75 Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ CD81/ HSP70 Not detected contaminants Albumin Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 100 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210383	2/2	Mus musculus	Serum	IAF	Mkrtchian, Soiuren	2022	75%
Study summary Full title Surgical Trauma in Mice Modifies the Content of Circulating Extracellular Vesicles All authors Souren Mkrtchian, Anette Ebberyd, Rosanne E. Veerman, María Méndez-Lago, Susanne Gabrielsson, Lars I. Eriksson, and Marta Gómez-Galán Journal Front Immunol Abstract Surgical interventions rapidly trigger a cascade of molecular, cellular, and neural signaling respon (show more...)Surgical interventions rapidly trigger a cascade of molecular, cellular, and neural signaling responses that ultimately reach remote organs, including the brain. Using a mouse model of orthopedic surgery, we have previously demonstrated hippocampal metabolic, structural, and functional changes associated with cognitive impairment. However, the nature of the underlying signals responsible for such periphery-to-brain communication remains hitherto elusive. Here we present the first exploratory study that tests the hypothesis of extracellular vesicles (EVs) as potential mediators carrying information from the injured tissue to the distal organs including the brain. The primary goal was to investigate whether the cargo of circulating EVs after surgery can undergo quantitative changes that could potentially trigger phenotypic modifications in the target tissues. EVs were isolated from the serum of the mice subjected to a tibia surgery after 6, 24, and 72 h, and the proteome and miRNAome were investigated using mass spectrometry and RNA-seq approaches. We found substantial differential expression of proteins and miRNAs starting at 6 h post-surgery and peaking at 24 h. Interestingly, one of the up-regulated proteins at 24 h was α-synuclein, a pathogenic hallmark of certain neurodegenerative syndromes. Analysis of miRNA target mRNA and corresponding biological pathways indicate the potential of post-surgery EVs to modify the extracellular matrix of the recipient cells and regulate metabolic processes including fatty acid metabolism. We conclude that surgery alters the cargo of circulating EVs in the blood, and our results suggest EVs as potential systemic signal carriers mediating remote effects of surgery on the brain. (hide) EV-METRIC 75% (98th 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 Immunoaffinity capture (non-commercial) Protein markers EV: CD63/ CD81/ HSP70 non-EV: Albumin/ Argonaute?2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Mus musculus Sample Type Serum Separation Method Immunoaffinity capture Selected surface protein(s) CD9/ CD63/ CD81 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) 0.75 Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ CD81/ HSP70 Detected contaminants Albumin Proteomics database Yes Characterization: RNA analysis RNA analysis Type (RT)?(q)PCR/ RNA?sequencing Database NGI Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 100 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210251	1/1	Sus scrofa	Follicular fluid	Exo-spin	Gad A	2022	75%
Study summary Full title Small-extracellular vesicles and their microRNA cargo from porcine follicular fluids: the potential association with oocyte quality. All authors Gad A, Murin M, Bartkova A, Kinterova V, Marcollova K, Laurincik J, Prochazka R Journal J Anim Sci Biotechnol Abstract Ovarian follicular fluids (FFs) contain several kinds of regulatory factors that maintain a suitable (show more...)Ovarian follicular fluids (FFs) contain several kinds of regulatory factors that maintain a suitable microenvironment for oocyte development. Extracellular vesicles (EVs) are among the factors that play essential roles in regulating follicle and oocyte development through their cargo molecules that include microRNAs (miRNAs). This study aimed to investigate small-EV (s-EV) miRNAs in porcine FFs and their potential association with oocyte quality. (hide) EV-METRIC 75% (95th 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 Follicular fluid 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 Commercial method Protein markers EV: Alix/ CD63/ TSG101 non-EV: ATP5A/ CytC Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Sus scrofa Sample Type Follicular fluid Separation Method Commercial kit Exo-spin Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) particles per milliliter of starting sample Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD63/ TSG101 Not detected contaminants ATP5A/ CytC Characterization: RNA analysis RNA analysis Type Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 135 EV concentration Yes Particle yield particles per milliliter of starting sample: 9.00e+9 EM EM-type Transmission EM Image type Close-up, Wide-field

	EV210237	1/2	Homo sapiens	Blood plasma	qEV	Gelibter S	2022	75%
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 75% (96th 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 Blood plasma 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 qEV Protein markers EV: Alix/ Flotillin-1/ Lamp1/ ANXA1 non-EV: GM130/ ApoE/ H3 Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) particles per milliliter of starting sample Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ Flotillin-1/ Lamp1/ ANXA1 Not detected contaminants GM130/ ApoE/ H3 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 EM EM-type Immuno-EM EM protein CD63 Image type Wide-field

	EV210165	1/2	Homo sapiens	THP-1	DG DC	Phu TA	2022	75%
Study summary Full title IL-4 polarized human macrophage exosomes control cardiometabolic inflammation and diabetes in obesity. All authors Phu TA, Ng M, Vu NK, Bouchareychas L, Raffai RL Journal Mol Ther Abstract Cardiometabolic disease is an increasing cause of morbidity and death in society. While M1-like macr (show more...)Cardiometabolic disease is an increasing cause of morbidity and death in society. While M1-like macrophages contribute to metabolic inflammation and insulin resistance, those polarized to an M2-like phenotype exert protective properties. Building on our observations reporting M2-like macrophage exosomes in atherosclerosis control, we tested whether they could serve to control inflammation in the liver and adipose tissue of obese mice. In thinking of clinical translation, we studied human THP-1 macrophages exposed to interleukin (IL)-4 as a source of exosomes (THP1-IL4-exo). Our findings show that THP1-IL4-exo polarized primary macrophages to an anti-inflammatory phenotype and reprogramed their energy metabolism by increasing levels of microRNA-21/99a/146b/378a (miR-21/99a/146b/378a) while reducing miR-33. This increased lipophagy, mitochondrial activity, and oxidative phosphorylation (OXPHOS). THP1-IL4-exo exerted a similar regulation of these miRs in cultured 3T3-L1 adipocytes. This enhanced insulin-dependent glucose uptake through increased peroxisome proliferator activated receptor gamma (PPARγ)-driven expression of GLUT4. It also increased levels of UCP1 and OXPHOS activity, which promoted lipophagy, mitochondrial activity, and beiging of 3T3-L1 adipocytes. Intraperitoneal infusions of THP1-IL4-exo into obese wild-type and Ldlr mice fed a Western high-fat diet reduced hematopoiesis and myelopoiesis, and favorably reprogramed inflammatory signaling and metabolism in circulating Ly6C monocytes. This also reduced leukocyte numbers and inflammatory activity in the circulation, aorta, adipose tissue, and the liver. Such treatments reduced hepatic steatosis and increased the beiging of white adipose tissue as revealed by increased UCP1 expression and OXPHOS activity that normalized blood insulin levels and improved glucose tolerance. Our findings support THP1-IL4-exo as a therapeutic approach to control cardiometabolic disease and diabetes in obesity. (hide) EV-METRIC 75% (96th 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 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 Density gradient Density cushion Protein markers EV: CD81/ CD63/ CD9 non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells THP-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell count 1,00E+06 Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 3 Orientation Top-down Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Density cushion Density medium Iodixanol Sample volume 98 Cushion volume 2 Density of the cushion 60% Centrifugation time 180 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81 Not detected contaminants Calnexin/ GM130 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 0,4 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 100 EV concentration Yes Particle yield as number of particles per million cells: 6.00e+0

	EV210165	2/2	Homo sapiens	THP-1	DG DC	Phu TA	2022	75%
Study summary Full title IL-4 polarized human macrophage exosomes control cardiometabolic inflammation and diabetes in obesity. All authors Phu TA, Ng M, Vu NK, Bouchareychas L, Raffai RL Journal Mol Ther Abstract Cardiometabolic disease is an increasing cause of morbidity and death in society. While M1-like macr (show more...)Cardiometabolic disease is an increasing cause of morbidity and death in society. While M1-like macrophages contribute to metabolic inflammation and insulin resistance, those polarized to an M2-like phenotype exert protective properties. Building on our observations reporting M2-like macrophage exosomes in atherosclerosis control, we tested whether they could serve to control inflammation in the liver and adipose tissue of obese mice. In thinking of clinical translation, we studied human THP-1 macrophages exposed to interleukin (IL)-4 as a source of exosomes (THP1-IL4-exo). Our findings show that THP1-IL4-exo polarized primary macrophages to an anti-inflammatory phenotype and reprogramed their energy metabolism by increasing levels of microRNA-21/99a/146b/378a (miR-21/99a/146b/378a) while reducing miR-33. This increased lipophagy, mitochondrial activity, and oxidative phosphorylation (OXPHOS). THP1-IL4-exo exerted a similar regulation of these miRs in cultured 3T3-L1 adipocytes. This enhanced insulin-dependent glucose uptake through increased peroxisome proliferator activated receptor gamma (PPARγ)-driven expression of GLUT4. It also increased levels of UCP1 and OXPHOS activity, which promoted lipophagy, mitochondrial activity, and beiging of 3T3-L1 adipocytes. Intraperitoneal infusions of THP1-IL4-exo into obese wild-type and Ldlr mice fed a Western high-fat diet reduced hematopoiesis and myelopoiesis, and favorably reprogramed inflammatory signaling and metabolism in circulating Ly6C monocytes. This also reduced leukocyte numbers and inflammatory activity in the circulation, aorta, adipose tissue, and the liver. Such treatments reduced hepatic steatosis and increased the beiging of white adipose tissue as revealed by increased UCP1 expression and OXPHOS activity that normalized blood insulin levels and improved glucose tolerance. Our findings support THP1-IL4-exo as a therapeutic approach to control cardiometabolic disease and diabetes in obesity. (hide) EV-METRIC 75% (96th 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 IL-4 cytokine treated 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 Density gradient Density cushion Protein markers EV: CD81/ CD63/ CD9 non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells THP-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell count 1,00E+06 Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 3 Orientation Top-down Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Density cushion Density medium Iodixanol Sample volume 98 Cushion volume 2 Density of the cushion 60% Centrifugation time 180 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81 Not detected contaminants Calnexin/ GM130 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 0,4 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 90,5 EV concentration Yes Particle yield as number of particles per million cells: 5.00e+0

	EV200029	1/2	Bos taurus	Bovine embryo culture medium	qEV	Pavani KC	2022	75%
Study summary Full title Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts. All authors Pavani KC, Meese T, Pascottini OB, Guan X, Lin X, Peelman L, Hamacher J, Van Nieuwerburgh F, Deforce D, Boel A, Heindryckx B, Tilleman K, Van Soom A, Gadella BM, Hendrix A, Smits K Journal Proc Natl Acad Sci U S A Abstract SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less l (show more...)SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less likely to occur in vitro for reasons unknown. Extracellular vesicles (EVs) are secreted by the embryo into the culture medium. Yet the role that embryonic EVs and their cargo microRNAs (miRNAs) play in blastocyst hatching has not been elucidated, partially due to the difficulties of isolating them from low amounts of culture medium. Here, we optimized EV-miRNA isolation from medium conditioned by individually cultured bovine embryos and subsequently showed that miR-378a-3p, which was up-regulated in EVs secreted by blastocysts, plays a crucial role in promoting blastocyst hatching. This demonstrates the regulatory effect of miR-378-3p on hatching, which is an established embryo quality parameter linked with implantation. (hide) EV-METRIC 75% (50th 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 Bovine embryo culture medium 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/ CD9 non-EV: AGO2/ APOA1 Proteomics no Show all info Study aim Biomarker Sample Species Bos taurus Sample Type Bovine embryo culture medium Separation Method Commercial kit qEV 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/ TSG101 Detected contaminants APOA1 Not detected contaminants AGO2 Characterization: RNA analysis RNA analysis Type RNA sequencing Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment Before RNAse type RNase A RNAse concentration 10 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 94.5±1.7 E08 EM EM-type Transmission-EM/ Cryo-EM Image type Close-up, Wide-field

	EV200029	2/2	Bos taurus	Bovine embryo culture medium	qEV	Pavani KC	2022	75%
Study summary Full title Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts. All authors Pavani KC, Meese T, Pascottini OB, Guan X, Lin X, Peelman L, Hamacher J, Van Nieuwerburgh F, Deforce D, Boel A, Heindryckx B, Tilleman K, Van Soom A, Gadella BM, Hendrix A, Smits K Journal Proc Natl Acad Sci U S A Abstract SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less l (show more...)SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less likely to occur in vitro for reasons unknown. Extracellular vesicles (EVs) are secreted by the embryo into the culture medium. Yet the role that embryonic EVs and their cargo microRNAs (miRNAs) play in blastocyst hatching has not been elucidated, partially due to the difficulties of isolating them from low amounts of culture medium. Here, we optimized EV-miRNA isolation from medium conditioned by individually cultured bovine embryos and subsequently showed that miR-378a-3p, which was up-regulated in EVs secreted by blastocysts, plays a crucial role in promoting blastocyst hatching. This demonstrates the regulatory effect of miR-378-3p on hatching, which is an established embryo quality parameter linked with implantation. (hide) EV-METRIC 75% (50th 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 Bovine embryo culture medium 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/ CD9 non-EV: AGO2/ APOA1 Proteomics no Show all info Study aim Biomarker Sample Species Bos taurus Sample Type Bovine embryo culture medium Separation Method Commercial kit qEV 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/ TSG101 Detected contaminants APOA1 Not detected contaminants AGO2 Characterization: RNA analysis RNA analysis Type RNA sequencing Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment Before RNAse type RNase A RNAse concentration 10 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 76.5±8.2 E08 EM EM-type Transmission-EM/ Cryo-EM Image type Close-up, Wide-field

	EV230967	1/1	Eimeria falciformis	NA	(d)(U)C DG UF Filtration	Olajide JS	2022	67%
Study summary Full title Eimeria falciformis secretes extracellular vesicles to modulate proinflammatory response during interaction with mouse intestinal epithelial cells. All authors Olajide JS, Xiong L, Yang S, Qu Z, Xu X, Yang B, Wang J, Liu B, Ma X, Cai J Journal Parasit Vectors Abstract Protozoan parasite secretions can be triggered by various modified media and diverse physicochemical (show more...)Protozoan parasite secretions can be triggered by various modified media and diverse physicochemical stressors. Equally, host-parasite interactions are known to co-opt the exchange and secretion of soluble biochemical components. Analysis of Eimeria falciformis sporozoite secretions in response to interaction with mouse intestinal epithelial cells (MIECs) may reveal parasite secretory motifs, protein composition and inflammatory activities of E. falciformis extracellular vesicles (EVs). (hide) EV-METRIC 67% (94th 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 Ultrafiltration Filtration Adj. k-factor 58515 (pelleting) / 58515 (washing) Protein markers EV: HSP70/ HSP90 non-EV: None Proteomics yes EV density (g/ml) 1.12-1.179 Show all info Study aim Biogenesis/cargo sorting/Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Eimeria falciformis Sample Type Cell culture supernatant EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 100 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: rotor type T-890 Pelleting: speed (g) 120,000 Pelleting: adjusted k-factor 58515 Wash: volume per pellet (ml) 1 Wash: time (min) 240 Wash: Rotor Type T-890 Wash: speed (g) 120000 Wash: adjusted k-factor 58515 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 5% Highest density fraction 50% Total gradient volume, incl. sample (mL) 7.4 Sample volume (mL) 1 Orientation Top-down Speed (g) 120,000 Duration (min) 1,200 Fraction volume (mL) 0.2 Fraction processing Centrifugation Pelleting: volume per fraction 0.2 Pelleting: speed (g) 120,000 Pelleting: adjusted k-factor 58515 Pelleting-wash: volume per pellet (mL) 0.1 Pelleting-wash: duration (min) 240 Pelleting-wash: speed (g) T-890 Filtration steps 0.2 or 0.22 ?m Ultra filtration Cut-off size (kDa) 10 Membrane type Polyethersulfone (PES) Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins HSP70 Not detected EV-associated proteins HSP90 Characterization: RNA analysis RNA analysis Type Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 246 ± 2 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV220403	1/2	Homo sapiens	Ishikawa	(d)(U)C DG	Fatmous M	2022	67%
Study summary Full title Endometrial small extracellular vesicles regulate human trophectodermal cell invasion by reprogramming the phosphoproteome landscape. All authors Fatmous M, Rai A, Poh QH, Salamonsen LA, Greening DW Journal Front Cell Dev Biol Abstract A series of cyclical events within the uterus are crucial for pregnancy establishment. These include (show more...)A series of cyclical events within the uterus are crucial for pregnancy establishment. These include endometrial regeneration following menses, under the influence of estrogen (proliferative phase), then endometrial differentiation driven by estrogen/progesterone (secretory phase), to provide a microenvironment enabling attachment of embryo (as a hatched blastocyst) to the endometrial epithelium. This is followed by invasion of trophectodermal cells (the outer layer of the blastocyst) into the endometrium tissue to facilitate intrauterine development. Small extracellular vesicles (sEVs) released by endometrial epithelial cells during the secretory phase have been shown to facilitate trophoblast invasion/ however, the molecular mechanisms that underline this process remain poorly understood. Here, we show that density gradient purified sEVs (1.06-1.11 g/ml, Alix and TSG101, ∼180 nm) from human endometrial epithelial cells (hormonally primed with estrogen and progesterone vs. estrogen alone) are readily internalized by a human trophectodermal stem cell line and promote their invasion into Matrigel matrix. Mass spectrometry-based proteome analysis revealed that sEVs reprogrammed trophectoderm cell proteome and their cell surface proteome (surfaceome) to support this invasive phenotype through upregulation of pro-invasive regulators associated with focal adhesions (NRP1, PTPRK, ROCK2, TEK), embryo implantation (FBLN1, NIBAN2, BSG), and kinase receptors (EPHB4/B2, ERBB2, STRAP). Kinase substrate prediction highlighted a central role of MAPK3 as an upstream kinase regulating target cell proteome reprogramming. Phosphoproteome analysis pinpointed upregulation of MAPK3 T204/T202 phosphosites in hTSCs following sEV delivery, and that their pharmacological inhibition significantly abrogated invasion. This study provides novel molecular insights into endometrial sEVs orchestrating trophoblast invasion, highlighting the microenvironmental regulation of hTSCs during embryo implantation. (hide) EV-METRIC 67% (94th 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 Secretory phase 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 Density gradient Protein markers EV: Alix/ TSG101 non-EV: None Proteomics yes EV density (g/ml) 1.06-1.11 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Ishikawa EV-harvesting Medium Serum free medium 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: rotor type SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 1 Wash: time (min) 60 Wash: Rotor Type SW 28 Wash: speed (g) 100000 Density gradient Type Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 3.6 Sample volume (mL) 0.2 Orientation Top-down Rotor type TLA-55 Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 0.3 Fraction processing Centrifugation Pelleting: volume per fraction 1 Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 0.05-0.1 Pelleting-wash: duration (min) 60 Pelleting-wash: speed (g) TLA-55 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/ TSG101 Proteomics database PRIDE Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 185.1 EV concentration Yes Particle yield particles per milliliter of starting sample: 8.21E+08

	EV220403	2/2	Homo sapiens	Ishikawa	(d)(U)C DG	Fatmous M	2022	67%
Study summary Full title Endometrial small extracellular vesicles regulate human trophectodermal cell invasion by reprogramming the phosphoproteome landscape. All authors Fatmous M, Rai A, Poh QH, Salamonsen LA, Greening DW Journal Front Cell Dev Biol Abstract A series of cyclical events within the uterus are crucial for pregnancy establishment. These include (show more...)A series of cyclical events within the uterus are crucial for pregnancy establishment. These include endometrial regeneration following menses, under the influence of estrogen (proliferative phase), then endometrial differentiation driven by estrogen/progesterone (secretory phase), to provide a microenvironment enabling attachment of embryo (as a hatched blastocyst) to the endometrial epithelium. This is followed by invasion of trophectodermal cells (the outer layer of the blastocyst) into the endometrium tissue to facilitate intrauterine development. Small extracellular vesicles (sEVs) released by endometrial epithelial cells during the secretory phase have been shown to facilitate trophoblast invasion/ however, the molecular mechanisms that underline this process remain poorly understood. Here, we show that density gradient purified sEVs (1.06-1.11 g/ml, Alix and TSG101, ∼180 nm) from human endometrial epithelial cells (hormonally primed with estrogen and progesterone vs. estrogen alone) are readily internalized by a human trophectodermal stem cell line and promote their invasion into Matrigel matrix. Mass spectrometry-based proteome analysis revealed that sEVs reprogrammed trophectoderm cell proteome and their cell surface proteome (surfaceome) to support this invasive phenotype through upregulation of pro-invasive regulators associated with focal adhesions (NRP1, PTPRK, ROCK2, TEK), embryo implantation (FBLN1, NIBAN2, BSG), and kinase receptors (EPHB4/B2, ERBB2, STRAP). Kinase substrate prediction highlighted a central role of MAPK3 as an upstream kinase regulating target cell proteome reprogramming. Phosphoproteome analysis pinpointed upregulation of MAPK3 T204/T202 phosphosites in hTSCs following sEV delivery, and that their pharmacological inhibition significantly abrogated invasion. This study provides novel molecular insights into endometrial sEVs orchestrating trophoblast invasion, highlighting the microenvironmental regulation of hTSCs during embryo implantation. (hide) EV-METRIC 67% (94th 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 Proliferactive phase 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 Density gradient Protein markers EV: Alix/ TSG101 non-EV: None Proteomics yes EV density (g/ml) 1.06-1.11 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Ishikawa EV-harvesting Medium Serum free medium 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: rotor type SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 1 Wash: time (min) 60 Wash: Rotor Type SW 28 Wash: speed (g) 100000 Density gradient Type Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 3.6 Sample volume (mL) 0.2 Orientation Top-down Rotor type TLA-55 Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 0.3 Fraction processing Centrifugation Pelleting: volume per fraction 1 Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 0.05-0.1 Pelleting-wash: duration (min) 60 Pelleting-wash: speed (g) TLA-55 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/ TSG101 Proteomics database PRIDE Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 180.6 EV concentration Yes Particle yield particles per milliliter of starting sample: 8.21E+08

	EV220293	1/1	Homo sapiens	Primary mesenchymal stromal cells	(d)(U)C DG	Nguyen, Vivian	2022	67%
Study summary Full title A human kidney and liver organoid-based multi-organ-on-a-chip model to study the therapeutic effects and biodistribution of mesenchymal stromal cell-derived extracellular vesicles All authors Vivian V T Nguyen, Shicheng Ye, Vasiliki Gkouzioti, Monique E van Wolferen, Fjodor Yousef Yengej, Dennis Melkert, Sofia Siti, Bart de Jong, Paul J Besseling, Bart Spee, Luc J W van der Laan, Reyk Horland, Marianne C Verhaar, Bas W M van Balkom Journal J Extracell Vesicles Abstract Mesenchymal stromal cell (MSC)-derived small extracellular vesicles (sEVs) show therapeutic potentia (show more...)Mesenchymal stromal cell (MSC)-derived small extracellular vesicles (sEVs) show therapeutic potential in multiple disease models, including kidney injury. Clinical translation of sEVs requires further preclinical and regulatory developments, including elucidation of the biodistribution and mode of action (MoA). Biodistribution can be determined using labelled sEVs in animal models which come with ethical concerns, are time-consuming and expensive, and may not well represent human physiology. We hypothesised that, based on developments in microfluidics and human organoid technology, in vitro multi-organ-on-a-chip (MOC) models allow us to study effects of sEVs in modelled human organs like kidney and liver in a semi-systemic manner. Human kidney- and liver organoids combined by microfluidic channels maintained physiological functions, and a kidney injury model was established using hydrogenperoxide. MSC-sEVs were isolated, and their size, density and potential contamination were analysed. These sEVs stimulated recovery of the renal epithelium after injury. Microscopic analysis shows increased accumulation of PKH67-labelled sEVs not only in injured kidney cells, but also in the unharmed liver organoids, compared to healthy control conditions. In conclusion, this new MOC model recapitulates therapeutic efficacy and biodistribution of MSC-sEVs as observed in animal models. Its human background allows for in-depth analysis of the MoA and identification of potential side effects. (hide) EV-METRIC 67% (94th 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 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 Density gradient Protein markers EV: Flotillin-1/ GAPDH non-EV: ATP5a/ Lamin A/C/ TOM20 Proteomics no EV density (g/ml) 1.13-1.14 Show all info Study aim Function/New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Primary mesenchymal stromal cells EV-harvesting Medium Serum free medium Cell count 10000000 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 32 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 4 Wash: time (min) 60 Wash: Rotor Type SW 60 Ti Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 0.25M Highest density fraction 2M Orientation Bottom-up Rotor type SW 60 Ti Speed (g) 190000 Duration (min) 960 Fraction volume (mL) 0.25 Fraction processing None Characterization: Protein analysis Protein Concentration Method NTA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin-1/ GAPDH Not detected contaminants ATP5a/ Lamin A/C/ TOM20 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 149 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.00E+10

	EV220091	1/8	Homo sapiens	HeLa	(d)(U)C SEC (non-commercial)	Visan, Kekoolani	2022	67%
Study summary Full title Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles All authors Kekoolani S. Visan, Richard J. Lobb, Sunyoung Ham, Luize G. Lima, Carlos Palma, Chai Pei Zhi Edna, Li-Ying Wu, Harsha Gowda, Keshava K. Datta, Gunter Hartel, Carlos Salomon, Andreas Möller Journal J Extracell Vesicles Abstract Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, progno (show more...)Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum-containing media. The majority of researchers use serum-containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non-sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA-MB-231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano-flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large-scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations. (hide) EV-METRIC 67% (94th 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 Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ HSP70/ TSG101 non-EV: Calnexin/ Albumin Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 1 Wash: time (min) 90 Wash: Rotor Type S55-A2 Wash: speed (g) 100,000 Size-exclusion chromatography Used for validation? Yes Total column volume (mL) 10 Sample volume/column (mL) 0.5 Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD9/ HSP70/ TSG101 Detected contaminants Albumin Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 117.7 EV concentration Yes Particle yield Total Particles: 1.63E+09 TRPS Report type Modus Reported size (nm) 105.3333333 EV concentration Yes Particle yield Total Particles: 8.91E+07 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210345	7/17	Homo sapiens	Expi293F	(d)(U)C	Osteikoetxea X	2022	67%
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 67% (94th 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 MysPalm-pMag-nMag-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/ syntenin-1/ B-actin Detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210345	9/17	Homo sapiens	Expi293F	(d)(U)C	Osteikoetxea X	2022	67%
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 67% (94th 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 MysPalm-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/ syntenin-1/ B-actin Detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210344	2/2	Homo sapiens	Saliva	(d)(U)C UF	Hofmann L	2022	67%
Study summary Full title Comparison of plasma- and saliva-derived exosomal miRNA profiles reveals diagnostic potential in head and neck cancer. All authors Hofmann L, Abou Kors T, Ezić J, Niesler B, Röth R, Ludwig S, Laban S, Schuler PJ, Hoffmann TK, Brunner C, Medyany V, Theodoraki MN Journal Front Cell Dev Biol Abstract Head and neck squamous cell carcinomas (HNSCC) lack tumor-specific biomarkers. Exosomes from HNSCC p (show more...)Head and neck squamous cell carcinomas (HNSCC) lack tumor-specific biomarkers. Exosomes from HNSCC patients carry immunomodulatory molecules, and correlate with clinical parameters. We compared miRNA profiles of plasma- and saliva-derived exosomes to reveal liquid biomarker candidates for HNSCC. Exosomes were isolated by differential ultracentrifugation from corresponding plasma and saliva samples from 11 HNSCC patients and five healthy donors (HD). Exosomal miRNA profiles, as determined by nCounter SPRINT technology, were analyzed regarding their diagnostic and prognostic potential, correlated to clinical data and integrated into network analysis. 119 miRNAs overlapped between plasma- and saliva-derived exosomes of HNSCC patients, from which 29 tumor-exclusive miRNAs, associated with , and signaling, were selected. By intra-correlation of tumor-exclusive miRNAs from plasma and saliva, top 10 miRNA candidates with the strongest correlation emerged as diagnostic panels to discriminate cancer and healthy as well as potentially prognostic panels for disease-free survival (DFS). Further, exosomal miRNAs were differentially represented in human papillomavirus (HPV) positive and negative as well as low and high stage disease. A plasma- and a saliva-derived panel of tumor-exclusive exosomal miRNAs hold great potential as liquid biopsy for discrimination between cancer and healthy as well as HPV status and disease stage. Exosomal miRNAs from both biofluids represent a promising tool for future biomarker studies, emphasizing the possibility to substitute plasma by less-invasive saliva collection. (hide) EV-METRIC 67% (89th 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 Saliva Sample origin Head and neck cancer 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 Ultrafiltration Protein markers EV: CD9/ CD63/ TSG101 non-EV: Grp94 Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Saliva 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 S55-A2 Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 1 Wash: time (min) 70 Wash: Rotor Type S55-A2 Wash: speed (g) 110000 Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Characterization: Protein analysis Protein Concentration Method BCA 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 CD9/ CD63/ TSG101 Not detected contaminants Grp94 Characterization: RNA analysis RNA analysis Type Microarray/ Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 101 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV210187	3/5	Homo sapiens	MOLM-14	(d)(U)C	Swatler, Julian	2022	67%
Study summary Full title 4-1BBL-containing leukemic extracellular vesicles promote immunosuppressive effector regulatory T cells All authors Julian Swatler, Laura Turos-Korgul, Marta Brewinska-Olchowik, Sara De Biasi, Wioleta Dudka, Bac Viet Le, Agata Kominek, Salwador Cyranowski, Paulina Pilanc, Elyas Mohammadi, Dominik Cysewski, Ewa Kozlowska, Wioleta Grabowska-Pyrzewicz, Urszula Wojda, Grzegorz W Basak, Jakub Mieczkowski, Tomasz Skorski 10 , Andrea Cossarizza 11 , Katarzyna Piwocka Journal Blood advances Abstract Chronic and acute myeloid leukemia (CML, AML) evade immune system surveillance and induce immunosupp (show more...)Chronic and acute myeloid leukemia (CML, AML) evade immune system surveillance and induce immunosuppression by expanding pro-leukemic Foxp3+ regulatory T cells (Tregs). High levels of immunosuppressive Tregs predict inferior response to chemotherapy, leukemia relapse and shorter survival. However, mechanisms that promote Tregs in myeloid leukemias remain largely unexplored. Here, we identify leukemic extracellular vesicles (EVs) as drivers of effector, pro-leukemic Tregs. Using mouse model of CML-like disease, we found that Rab27a-dependent secretion of leukemic EVs promoted leukemia engraftment, which was associated with higher abundance of activated, immunosuppressive Tregs. Leukemic EVs attenuated mTOR-S6 and activated STAT5 signaling, as well as evoked significant transcriptomic changes in Tregs. We further identified specific effector signature of Tregs promoted by leukemic EVs. Leukemic EVs-driven Tregs were characterized by elevated expression of effector/tumor Treg markers CD39, CCR8, CD30, TNFR2, CCR4, TIGIT, IL21R and included two distinct, effector Treg (eTreg) subsets - CD30+CCR8hiTNFR2hi eTreg1 and CD39+TIGIThi eTreg2. Finally, we showed that costimulatory ligand 4-1BBL/CD137L, shuttled by leukemic EVs, promoted suppressive activity and effector phenotype of Tregs by regulating expression of receptors such as CD30 and TNFR2. Collectively, our work highlights the role of leukemic extracellular vesicles in stimulation of immunosuppressive regulatory T cells and leukemia growth. We postulate that targeting of Rab27a-dependent secretion of leukemic EVs may be a viable therapeutic approach in myeloid neoplasms. (hide) EV-METRIC 67% (94th 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 Protein markers EV: TSG101 non-EV: APOA1/ GM130 Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MOLM-14 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 98 Cell count 2.80E+08 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: time(min) 90 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 60 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins TSG101 Not detected contaminants APOA1/ GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 95 EV concentration Yes Particle yield Yes, as number of particles per million cells 5.00E+07 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210168	1/3	Homo sapiens	Blood plasma	qEV IAF	Newman, Lauren	2022	67%
Study summary Full title Selective Isolation of Liver-Derived Extracellular Vesicles Redefines Performance of miRNA Biomarkers for Non-Alcoholic Fatty Liver Disease All authors Lauren A Newman, Zivile Useckaite, Jillian Johnson, Michael J Sorich, Ashley M Hopkins, Andrew Rowland Journal biomedicines Abstract Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagn (show more...)Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagnosis of the progressive form, non-alcoholic steatohepatitis (NASH), requires liver biopsy, which is highly invasive and unsuited to early disease or tracking changes. Inadequate performance of current minimally invasive tools is a critical barrier to managing NAFLD burden. Altered circulating miRNA profiles show potential for minimally invasive tracking of NAFLD. The selective isolation of the circulating extracellular vesicle subset that originates from hepatocytes presents an important opportunity for improving the performance of miRNA biomarkers of liver disease. The expressions of miR-122, -192, and -128-3p were quantified in total cell-free RNA, global EVs, and liver-specific EVs from control, NAFL, and NASH subjects. In ASGR1+ EVs, each miR biomarker trended positively with disease severity and expression was significantly higher in NASH subjects compared with controls. The c-statistic defining the performance of ASGR1+ EV derived miRNAs was invariably >0.78. This trend was not observed in the alternative sources. This study demonstrates the capacity for liver-specific isolation to transform the performance of EV-derived miRNA biomarkers for NAFLD, robustly distinguishing patients with NAFL and NASH. (hide) EV-METRIC 67% (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 Blood plasma 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 Immunoaffinity capture (non-commercial) Protein markers EV: None non-EV: albumin/ calnexin Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Immunoaffinity capture Selected surface protein(s) ASGR1 Characterization: Protein analysis Protein Concentration Method microBCA Proteomics database No Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 102.9 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.17E+11 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210168	2/3	Homo sapiens	Blood plasma	qEV IAF	Newman, Lauren	2022	67%
Study summary Full title Selective Isolation of Liver-Derived Extracellular Vesicles Redefines Performance of miRNA Biomarkers for Non-Alcoholic Fatty Liver Disease All authors Lauren A Newman, Zivile Useckaite, Jillian Johnson, Michael J Sorich, Ashley M Hopkins, Andrew Rowland Journal biomedicines Abstract Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagn (show more...)Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagnosis of the progressive form, non-alcoholic steatohepatitis (NASH), requires liver biopsy, which is highly invasive and unsuited to early disease or tracking changes. Inadequate performance of current minimally invasive tools is a critical barrier to managing NAFLD burden. Altered circulating miRNA profiles show potential for minimally invasive tracking of NAFLD. The selective isolation of the circulating extracellular vesicle subset that originates from hepatocytes presents an important opportunity for improving the performance of miRNA biomarkers of liver disease. The expressions of miR-122, -192, and -128-3p were quantified in total cell-free RNA, global EVs, and liver-specific EVs from control, NAFL, and NASH subjects. In ASGR1+ EVs, each miR biomarker trended positively with disease severity and expression was significantly higher in NASH subjects compared with controls. The c-statistic defining the performance of ASGR1+ EV derived miRNAs was invariably >0.78. This trend was not observed in the alternative sources. This study demonstrates the capacity for liver-specific isolation to transform the performance of EV-derived miRNA biomarkers for NAFLD, robustly distinguishing patients with NAFL and NASH. (hide) EV-METRIC 67% (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 Blood plasma Sample origin NAFL 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 Immunoaffinity capture (non-commercial) Protein markers EV: None non-EV: albumin/ calnexin Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Immunoaffinity capture Selected surface protein(s) ASGR1 Characterization: Protein analysis Protein Concentration Method microBCA Proteomics database No Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 113.3 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2.34E+11 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210168	3/3	Homo sapiens	Blood plasma	qEV IAF	Newman, Lauren	2022	67%
Study summary Full title Selective Isolation of Liver-Derived Extracellular Vesicles Redefines Performance of miRNA Biomarkers for Non-Alcoholic Fatty Liver Disease All authors Lauren A Newman, Zivile Useckaite, Jillian Johnson, Michael J Sorich, Ashley M Hopkins, Andrew Rowland Journal biomedicines Abstract Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagn (show more...)Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagnosis of the progressive form, non-alcoholic steatohepatitis (NASH), requires liver biopsy, which is highly invasive and unsuited to early disease or tracking changes. Inadequate performance of current minimally invasive tools is a critical barrier to managing NAFLD burden. Altered circulating miRNA profiles show potential for minimally invasive tracking of NAFLD. The selective isolation of the circulating extracellular vesicle subset that originates from hepatocytes presents an important opportunity for improving the performance of miRNA biomarkers of liver disease. The expressions of miR-122, -192, and -128-3p were quantified in total cell-free RNA, global EVs, and liver-specific EVs from control, NAFL, and NASH subjects. In ASGR1+ EVs, each miR biomarker trended positively with disease severity and expression was significantly higher in NASH subjects compared with controls. The c-statistic defining the performance of ASGR1+ EV derived miRNAs was invariably >0.78. This trend was not observed in the alternative sources. This study demonstrates the capacity for liver-specific isolation to transform the performance of EV-derived miRNA biomarkers for NAFLD, robustly distinguishing patients with NAFL and NASH. (hide) EV-METRIC 67% (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 Blood plasma Sample origin NASH 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 Immunoaffinity capture (non-commercial) Protein markers EV: None non-EV: albumin/ calnexin Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Immunoaffinity capture Selected surface protein(s) ASGR1 Characterization: Protein analysis Protein Concentration Method microBCA Proteomics database No Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 110.1 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2.73E+11 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210076	1/6	Rattus norvegicus	Urine	(d)(U)C Filtration	Hinzman CP	2022	67%
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 67% (94th 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 (Differential) (ultra)centrifugation Filtration 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 (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) 120.000 Filtration steps 0.22µm or 0.2µm 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/ CD63/ EpCAM/ ANXA5/ TSG101/ Alix/ CD81 Detected contaminants GM130 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 135 EV concentration Yes EM EM-type Cryo-EM Image type Close-up

	EV210076	4/6	Rattus norvegicus	Urine	(d)(U)C Filtration	Hinzman CP	2022	67%
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 67% (94th 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 (Differential) (ultra)centrifugation Filtration 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 (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) 120.000 Filtration steps 0.22µm or 0.2µm 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/ EpCam/ ANXA5/ CD63/ TSG101/ CD81 Detected contaminants GM130 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 134.9 EV concentration Yes EM EM-type Cryo-EM Image type Close-up

	EV210024	8/12	Homo sapiens	Glioblastoma Stem-like cells (GSC)	(d)(U)C DG	André-Grégoire, Gwennan	2022	67%
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 67% (94th 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 Density gradient Protein markers EV: Alix/ CD63 non-EV: GM130 Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Glioblastoma Stem-like cells (GSC) EV-harvesting Medium Serum free medium Cell count 5.00E+08 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 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40 Total gradient volume, incl. sample (mL) 11.5 Sample volume (mL) 0.5 Orientation Top-down Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 11 Pelleting: duration (min) 120 Pelleting: rotor type SW 41 Ti Pelleting: 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 CD63/ Alix Not detected contaminants GM130 ELISA Antibody details provided? Yes Lysis buffer provided? Yes 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 Report size (nm) 100

	EV210002	2/3	Mus musculus	dissociated tissues	(d)(U)C	Delcorte, Ophélie	2022	67%
Study summary Full title BRAF V600E Induction in Thyrocytes Triggers Important Changes in the miRNAs Content and the Populations of Extracellular Vesicles Released in Thyroid Tumor Microenvironment All authors Ophélie Delcorte, Catherine Spourquet, Pascale Lemoine, Jonathan Degosserie, Patrick Van Der Smissen, Nicolas Dauguet, Axelle Loriot, Jeffrey A Knauf, Laurent Gatto, Etienne Marbaix, James A Fagin, Christophe E Pierreux Journal Biomediscines Abstract Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recur (show more...)Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recurrences still challenge clinicians. New perspectives to overcome these issues could come from the study of extracellular vesicle (EV) populations and content. Here, we aimed to elucidate the heterogeneity of EVs circulating in the tumor and the changes in their microRNA content during cancer progression. Using a mouse model expressing BRAFV600E, we isolated and characterized EVs from thyroid tissue by ultracentrifugations and elucidated their microRNA content by small RNA sequencing. The cellular origin of EVs was investigated by ExoView and that of deregulated EV-microRNA by qPCR on FACS-sorted cell populations. We found that PTC released more EVs bearing epithelial and immune markers, as compared to the healthy thyroid, so that changes in EV-microRNAs abundance were mainly due to their deregulated expression in thyrocytes. Altogether, our work provides a full description of in vivo-derived EVs produced by, and within, normal and cancerous thyroid. We elucidated the global EV-microRNAs signature, the dynamic loading of microRNAs in EVs upon BRAFV600E induction, and their cellular origin. Finally, we propose that thyroid tumor-derived EV-microRNAs could support the establishment of a permissive immune microenvironment. (hide) EV-METRIC 67% (33rd 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 dissociated tissues Sample origin BRAFV600E-induced tissue (Doxycyxline 2*24h) 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 No extra separation steps Protein markers EV: Alix/ CD63/ Flotillin1/ CD9/ CD81 non-EV: Calnexin/ PDI Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Mus musculus Sample Type dissociated tissues Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 80 Ti Pelleting: speed (g) 150000 Wash: volume per pellet (ml) 7 Wash: time (min) 90 Wash: Rotor Type Type 80 Ti Wash: speed (g) 150000 Other Name other separation method No extra separation steps Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ CD9/ CD63/ Alix/ CD81 Not detected contaminants Calnexin/ PDI Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing Database Yes Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment Before RNAse type RNase A RNAse concentration 0,01 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes Particle yield Per dissociated thyroid;Yes, other: 3,30E+09 EM EM-type Transmission-EM Image type Close-up

	EV210002	3/3	Mus musculus	dissociated tissues	(d)(U)C	Delcorte, Ophélie	2022	67%
Study summary Full title BRAF V600E Induction in Thyrocytes Triggers Important Changes in the miRNAs Content and the Populations of Extracellular Vesicles Released in Thyroid Tumor Microenvironment All authors Ophélie Delcorte, Catherine Spourquet, Pascale Lemoine, Jonathan Degosserie, Patrick Van Der Smissen, Nicolas Dauguet, Axelle Loriot, Jeffrey A Knauf, Laurent Gatto, Etienne Marbaix, James A Fagin, Christophe E Pierreux Journal Biomediscines Abstract Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recur (show more...)Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recurrences still challenge clinicians. New perspectives to overcome these issues could come from the study of extracellular vesicle (EV) populations and content. Here, we aimed to elucidate the heterogeneity of EVs circulating in the tumor and the changes in their microRNA content during cancer progression. Using a mouse model expressing BRAFV600E, we isolated and characterized EVs from thyroid tissue by ultracentrifugations and elucidated their microRNA content by small RNA sequencing. The cellular origin of EVs was investigated by ExoView and that of deregulated EV-microRNA by qPCR on FACS-sorted cell populations. We found that PTC released more EVs bearing epithelial and immune markers, as compared to the healthy thyroid, so that changes in EV-microRNAs abundance were mainly due to their deregulated expression in thyrocytes. Altogether, our work provides a full description of in vivo-derived EVs produced by, and within, normal and cancerous thyroid. We elucidated the global EV-microRNAs signature, the dynamic loading of microRNAs in EVs upon BRAFV600E induction, and their cellular origin. Finally, we propose that thyroid tumor-derived EV-microRNAs could support the establishment of a permissive immune microenvironment. (hide) EV-METRIC 67% (33rd 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 dissociated tissues Sample origin BRAFV600E-induced tissue (Doxycyxline4*24h) 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 No extra separation steps Protein markers EV: Alix/ CD63/ Flotillin1/ CD9/ CD81 non-EV: Calnexin/ PDI Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Mus musculus Sample Type dissociated tissues Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 80 Ti Pelleting: speed (g) 150000 Wash: volume per pellet (ml) 7 Wash: time (min) 90 Wash: Rotor Type Type 80 Ti Wash: speed (g) 150000 Other Name other separation method No extra separation steps Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ Alix/ CD9/ CD63/ CD81 Not detected contaminants Calnexin/ PDI Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing Database Yes Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment Before RNAse type RNase A RNAse concentration 0,01 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes Particle yield Per dissociated thyroid;Yes, other: 1,20E+10 EM EM-type Transmission-EM Image type Close-up

	EV200174	1/2	Bos taurus	1% skim milk	(d)(U)C	Sukreet, Sonal	2022	67%
Study summary Full title Ultrasonication of Milk Decreases the Content of Exosomes and MicroRNAs in an Exosome-defined Rodent Diet All authors Sonal Sukreet, Camila Pereira Braga, Thuy T An, Jiri Adamec, Juan Cui, Janos Zempleni Journal J Nutr Abstract Background: Bovine milk exosomes (BMEs) harbor regulatory proteins, lipids and microRNAs. Consumptio (show more...)Background: Bovine milk exosomes (BMEs) harbor regulatory proteins, lipids and microRNAs. Consumption of an exosome and RNA-depleted (ERD) diet elicited phenotypes compared to controls fed an exosome and RNA-sufficient (ERS) diet in mice. All other ingredients were identical in the diets. ERD and ERS diets were prepared by substituting ultrasonicated and non-ultrasonicated milk, respectively, for casein in the AIN-93G formulation. Objective: The objective of this study was to assess the effect of ultrasonication of milk on exosome content and bioavailability, and cargo content. Methods: Bovine milk was ultrasonicated and exosomes were isolated by ultracentrifugation (USE); controls were not ultrasonicated (NSE). Exosome count, size and morphology were assessed using a nanoparticle tracker and electron microscopy. RNAs, lipids and proteins were analyzed by RNA-sequencing and mass spectrometry. Intestinal transport, bioavailability and distribution were measured by using fluorophore-labeled USEs and NSEs in Caco-2 cells, FHs 74 Int cells and C57BL/6J mice (n = 3; age 6 - 8 weeks). Results: The exosome count was 76 ± 22% lower in USE compared to NSE (P < 0.05). Ultrasonication caused a degradation of up to 100% of microRNAs. USE and NSE contained 145 and 332 unique lipid signatures, respectively (P < 0.05). We detected total of 525 and 484 proteins in USE and NSE. The uptake of USEs decreased by 46 ± 30% and 40 ± 27% compared to NSEs in Caco-2 and FHs 74 Int cells, respectively (P < 0.05). The hepatic accumulation of USEs was 48% ± 28% lower than the accumulation of NSEs in mice (P < 0.05). Conclusions: Ultrasonication of milk depletes bioavailable BMEs in studies of Caco-2 cells, FHs 74 Int cells and C57BL/6J mice and causes a near-complete degradation of microRNA cargos. (hide) EV-METRIC 67% (66th 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 No extra separation steps Protein markers EV: TSG101/ Alix/ CD63/ CD9/ CD81 non-EV: Beta-integrin/ lactalbumin/ Histone-H3 Proteomics yes Show all info Study aim Function/New methodological development/Identification of content (omics approaches)/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) 30 mL Wash: time (min) 90 Wash: Rotor Type Fiberlite-F37L-8x100 rotor Wash: speed (g) 120,000 Other Name other separation method No extra separation steps Characterization: Protein analysis Protein Concentration Method BCA;Qubit Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ TSG101/ Alix/ CD81 Detected contaminants lactalbumin Not detected contaminants Beta-integrin/ Histone-H3 Proteomics database No Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNA sequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 117+/-44 EV concentration Yes Particle yield Yes, per milliliter of starting sample 5.90E+11 EM EM-type Transmission-EM/ Scanning-EM Image type Close-up, Wide-field

	EV200174	2/2	Bos taurus	1% skim milk	(d)(U)C	Sukreet, Sonal	2022	67%
Study summary Full title Ultrasonication of Milk Decreases the Content of Exosomes and MicroRNAs in an Exosome-defined Rodent Diet All authors Sonal Sukreet, Camila Pereira Braga, Thuy T An, Jiri Adamec, Juan Cui, Janos Zempleni Journal J Nutr Abstract Background: Bovine milk exosomes (BMEs) harbor regulatory proteins, lipids and microRNAs. Consumptio (show more...)Background: Bovine milk exosomes (BMEs) harbor regulatory proteins, lipids and microRNAs. Consumption of an exosome and RNA-depleted (ERD) diet elicited phenotypes compared to controls fed an exosome and RNA-sufficient (ERS) diet in mice. All other ingredients were identical in the diets. ERD and ERS diets were prepared by substituting ultrasonicated and non-ultrasonicated milk, respectively, for casein in the AIN-93G formulation. Objective: The objective of this study was to assess the effect of ultrasonication of milk on exosome content and bioavailability, and cargo content. Methods: Bovine milk was ultrasonicated and exosomes were isolated by ultracentrifugation (USE); controls were not ultrasonicated (NSE). Exosome count, size and morphology were assessed using a nanoparticle tracker and electron microscopy. RNAs, lipids and proteins were analyzed by RNA-sequencing and mass spectrometry. Intestinal transport, bioavailability and distribution were measured by using fluorophore-labeled USEs and NSEs in Caco-2 cells, FHs 74 Int cells and C57BL/6J mice (n = 3; age 6 - 8 weeks). Results: The exosome count was 76 ± 22% lower in USE compared to NSE (P < 0.05). Ultrasonication caused a degradation of up to 100% of microRNAs. USE and NSE contained 145 and 332 unique lipid signatures, respectively (P < 0.05). We detected total of 525 and 484 proteins in USE and NSE. The uptake of USEs decreased by 46 ± 30% and 40 ± 27% compared to NSEs in Caco-2 and FHs 74 Int cells, respectively (P < 0.05). The hepatic accumulation of USEs was 48% ± 28% lower than the accumulation of NSEs in mice (P < 0.05). Conclusions: Ultrasonication of milk depletes bioavailable BMEs in studies of Caco-2 cells, FHs 74 Int cells and C57BL/6J mice and causes a near-complete degradation of microRNA cargos. (hide) EV-METRIC 67% (66th 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 Sonicated milk 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: CD81/ Alix/ TSG101/ CD63/ CD9 non-EV: Beta-integrin/ lactalbumin/ Histone H3 Proteomics yes Show all info Study aim Function/New methodological development/Identification of content (omics approaches)/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) 30 mL Wash: time (min) 90 Wash: Rotor Type Fiberlite-F37L-8x100 rotor Wash: speed (g) 120,000 Characterization: Protein analysis Protein Concentration Method BCA;Qubit Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ TSG101 Not detected EV-associated proteins CD81/ Alix Detected contaminants lactalbumin Not detected contaminants Beta-integrin/ Histone H3 Proteomics database No Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 218+/-99 EV concentration Yes Particle yield Yes, per milliliter of starting sample 1.40E+11 EM EM-type Transmission-EM/ Scanning-EM Image type Close-up, Wide-field

	EV200126	1/6	Homo sapiens	Brain tissue	(d)(U)C Filtration	Huang Y	2022	67%
Study summary Full title Brain Tissue-Derived Extracellular Vesicles in Alzheimer's Disease Display Altered Key Protein Levels Including Cell Type-Specific Markers. All authors Huang Y, Driedonks TAP, Cheng L, Rajapaksha H, Routenberg DA, Nagaraj R, Redding J, Arab T, Powell BH, Pletniková O, Troncoso JC, Zheng L, Hill AF, Mahairaki V, Witwer KW Journal J Alzheimers Dis Abstract Brain tissue-derived extracellular vesicles (bdEVs) play neurodegenerative and protective roles, inc (show more...)Brain tissue-derived extracellular vesicles (bdEVs) play neurodegenerative and protective roles, including in Alzheimer's disease (AD). Extracellular vesicles (EVs) may also leave the brain to betray the state of the CNS in the periphery. Only a few studies have profiled the proteome of bdEVs and source brain tissue. Additionally, studies focusing on bdEV cell type-specific surface markers are rare. (hide) EV-METRIC 67% (81st 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 Brain tissue 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 Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Brain tissue 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 Yes Pelleting: rotor type AH-650 Pelleting: speed (g) 10000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Proteomics database No Characterization: RNA analysis RNA analysis Type RNA sequencing Database Yes: Gene Expression Omnibus Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type nanoFCM flow nanoAnalyzer Hardware adjustment Dedicated flow cytometer for nanosized particles Calibration bead size 0.068/ 0.091/ 0.113/ 0.151 Report type Size range/distribution Reported size (nm) 40-145 EV concentration Yes Particle yield other:/ number of particles per 100 mg brain tissue: 1.14E+09 EM EM-type Transmission-EM Image type Wide-field Report size (nm) Not reported

	EV200126	2/6	Homo sapiens	Brain tissue	(d)(U)C qEV UF Filtration	Huang Y	2022	67%
Study summary Full title Brain Tissue-Derived Extracellular Vesicles in Alzheimer's Disease Display Altered Key Protein Levels Including Cell Type-Specific Markers. All authors Huang Y, Driedonks TAP, Cheng L, Rajapaksha H, Routenberg DA, Nagaraj R, Redding J, Arab T, Powell BH, Pletniková O, Troncoso JC, Zheng L, Hill AF, Mahairaki V, Witwer KW Journal J Alzheimers Dis Abstract Brain tissue-derived extracellular vesicles (bdEVs) play neurodegenerative and protective roles, inc (show more...)Brain tissue-derived extracellular vesicles (bdEVs) play neurodegenerative and protective roles, including in Alzheimer's disease (AD). Extracellular vesicles (EVs) may also leave the brain to betray the state of the CNS in the periphery. Only a few studies have profiled the proteome of bdEVs and source brain tissue. Additionally, studies focusing on bdEV cell type-specific surface markers are rare. (hide) EV-METRIC 67% (81st 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 Brain tissue 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 Commercial method Ultrafiltration Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Brain tissue 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 TH-641 Pelleting: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Proteomics database No Characterization: RNA analysis RNA analysis Type RNAsequencing Database Yes: Gene Expression Omnibus Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type nanoFCM flow nanoAnalyzer Hardware adjustment Dedicated flow cytometer for nanosized particles Calibration bead size 0.068/ 0.091/ 0.113/ 0.151 Report type Size range/distribution Reported size (nm) 40-145 EV concentration Yes Particle yield other:/ number of particles per 100 mg brain tissue: 3.36E+09 EM EM-type Transmission-EM Image type Wide-field Report size (nm) Not reported

	EV200126	3/6	Homo sapiens	Brain tissue	(d)(U)C qEV UF Filtration	Huang Y	2022	67%
Study summary Full title Brain Tissue-Derived Extracellular Vesicles in Alzheimer's Disease Display Altered Key Protein Levels Including Cell Type-Specific Markers. All authors Huang Y, Driedonks TAP, Cheng L, Rajapaksha H, Routenberg DA, Nagaraj R, Redding J, Arab T, Powell BH, Pletniková O, Troncoso JC, Zheng L, Hill AF, Mahairaki V, Witwer KW Journal J Alzheimers Dis Abstract Brain tissue-derived extracellular vesicles (bdEVs) play neurodegenerative and protective roles, inc (show more...)Brain tissue-derived extracellular vesicles (bdEVs) play neurodegenerative and protective roles, including in Alzheimer's disease (AD). Extracellular vesicles (EVs) may also leave the brain to betray the state of the CNS in the periphery. Only a few studies have profiled the proteome of bdEVs and source brain tissue. Additionally, studies focusing on bdEV cell type-specific surface markers are rare. (hide) EV-METRIC 67% (81st 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 Brain tissue Sample origin Alzheimer 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 Commercial method Ultrafiltration Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Brain tissue 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 TH-641 Pelleting: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Proteomics database No Characterization: RNA analysis RNA analysis Type RNAsequencing Database Yes: Gene Expression Omnibus Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type nanoFCM flow nanoAnalyzer Hardware adjustment Dedicated flow cytometer for nanosized particles Calibration bead size 0.068/ 0.091/ 0.113/ 0.151 Report type Size range/distribution Reported size (nm) 40-145 EV concentration Yes Particle yield other:/ number of particles per 100 mg brain tissue: 2.25E+09 EM EM-type Transmission-EM Image type Wide-field Report size (nm) Not reported

	EV200126	4/6	Homo sapiens	Brain tissue	(d)(U)C Filtration	Huang Y	2022	67%
Study summary Full title Brain Tissue-Derived Extracellular Vesicles in Alzheimer's Disease Display Altered Key Protein Levels Including Cell Type-Specific Markers. All authors Huang Y, Driedonks TAP, Cheng L, Rajapaksha H, Routenberg DA, Nagaraj R, Redding J, Arab T, Powell BH, Pletniková O, Troncoso JC, Zheng L, Hill AF, Mahairaki V, Witwer KW Journal J Alzheimers Dis Abstract Brain tissue-derived extracellular vesicles (bdEVs) play neurodegenerative and protective roles, inc (show more...)Brain tissue-derived extracellular vesicles (bdEVs) play neurodegenerative and protective roles, including in Alzheimer's disease (AD). Extracellular vesicles (EVs) may also leave the brain to betray the state of the CNS in the periphery. Only a few studies have profiled the proteome of bdEVs and source brain tissue. Additionally, studies focusing on bdEV cell type-specific surface markers are rare. (hide) EV-METRIC 67% (81st 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 Brain tissue Sample origin Alzheimer 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 Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Brain tissue 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 Yes Pelleting: rotor type AH-650 Pelleting: speed (g) 10000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Proteomics database No Characterization: RNA analysis RNA analysis Type RNAsequencing Database Yes: Gene Expression Omnibus Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type nanoFCM flow nanoAnalyzer Hardware adjustment Dedicated flow cytometer for nanosized particles Calibration bead size 0.068/ 0.091/ 0.113/ 0.151 Report type Size range/distribution Reported size (nm) 40-145 EV concentration Yes Particle yield other:/ number of particles per 100 mg brain tissue: 1.14E+09 EM EM-type Transmission-EM Image type Wide-field Report size (nm) Not reported

	EV200126	5/6	Homo sapiens	Brain tissue	(d)(U)C qEV UF Filtration	Huang Y	2022	67%
Study summary Full title Brain Tissue-Derived Extracellular Vesicles in Alzheimer's Disease Display Altered Key Protein Levels Including Cell Type-Specific Markers. All authors Huang Y, Driedonks TAP, Cheng L, Rajapaksha H, Routenberg DA, Nagaraj R, Redding J, Arab T, Powell BH, Pletniková O, Troncoso JC, Zheng L, Hill AF, Mahairaki V, Witwer KW Journal J Alzheimers Dis Abstract Brain tissue-derived extracellular vesicles (bdEVs) play neurodegenerative and protective roles, inc (show more...)Brain tissue-derived extracellular vesicles (bdEVs) play neurodegenerative and protective roles, including in Alzheimer's disease (AD). Extracellular vesicles (EVs) may also leave the brain to betray the state of the CNS in the periphery. Only a few studies have profiled the proteome of bdEVs and source brain tissue. Additionally, studies focusing on bdEV cell type-specific surface markers are rare. (hide) EV-METRIC 67% (81st 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 Brain tissue Sample origin APOE genotype 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 Commercial method Ultrafiltration Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Brain tissue 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 TH-641 Pelleting: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Proteomics database No Characterization: RNA analysis RNA analysis Type RNAsequencing Database Yes: Gene Expression Omnibus Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type nanoFCM flow nanoAnalyzer Hardware adjustment Dedicated flow cytometer for nanosized particles Calibration bead size 0.068/ 0.091/ 0.113/ 0.151 Report type Size range/distribution Reported size (nm) 40-145 EV concentration Yes Particle yield other:/ number of particles per 100 mg brain tissue: 2.25E+09 EM EM-type Transmission-EM Image type Wide-field Report size (nm) Not reported

	EV200126	6/6	Homo sapiens	Brain tissue	(d)(U)C Filtration	Huang Y	2022	67%
Study summary Full title Brain Tissue-Derived Extracellular Vesicles in Alzheimer's Disease Display Altered Key Protein Levels Including Cell Type-Specific Markers. All authors Huang Y, Driedonks TAP, Cheng L, Rajapaksha H, Routenberg DA, Nagaraj R, Redding J, Arab T, Powell BH, Pletniková O, Troncoso JC, Zheng L, Hill AF, Mahairaki V, Witwer KW Journal J Alzheimers Dis Abstract Brain tissue-derived extracellular vesicles (bdEVs) play neurodegenerative and protective roles, inc (show more...)Brain tissue-derived extracellular vesicles (bdEVs) play neurodegenerative and protective roles, including in Alzheimer's disease (AD). Extracellular vesicles (EVs) may also leave the brain to betray the state of the CNS in the periphery. Only a few studies have profiled the proteome of bdEVs and source brain tissue. Additionally, studies focusing on bdEV cell type-specific surface markers are rare. (hide) EV-METRIC 67% (81st 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 Brain tissue Sample origin APOE genotype 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 Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Brain tissue 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 Yes Pelleting: rotor type AH-650 Pelleting: speed (g) 10000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Proteomics database No Characterization: RNA analysis RNA analysis Type RNAsequencing Database Yes: Gene Expression Omnibus Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type nanoFCM flow nanoAnalyzer Hardware adjustment Dedicated flow cytometer for nanosized particles Calibration bead size 0.068/ 0.091/ 0.113/ 0.151 Report type Size range/distribution Reported size (nm) 40-145 EV concentration Yes Particle yield other:/ number of particles per 100 mg brain tissue: 1.14E+09 EM EM-type Transmission-EM Image type Wide-field Report size (nm) Not reported

	EV200112	1/2	Homo sapiens	HUVEC	(d)(U)C	Giraud, Romain	2022	67%
Study summary Full title Tracking Radiolabeled Endothelial Microvesicles Predicts Their Therapeutic Efficacy: A Proof-of-Concept Study in Peripheral Ischemia Mouse Model Using SPECT/CT Imaging All authors Romain Giraud, Anaïs Moyon, Stéphanie Simoncini, Anne-Claire Duchez, Vincent Nail, Corinne Chareyre, Ahlem Bouhlel, Laure Balasse, Samantha Fernandez, Loris Vallier, Guillaume Hache, Florence Sabatier, Françoise Dignat-George, Romaric Lacroix, Benjamin Guillet, Philippe Garrigue Journal Pharmaceutics Abstract Microvesicles, so-called endothelial large extracellular vesicles (LEVs), are of great interest as b (show more...)Microvesicles, so-called endothelial large extracellular vesicles (LEVs), are of great interest as biological markers and cell-free biotherapies in cardiovascular and oncologic diseases. However, their therapeutic perspectives remain limited due to the lack of reliable data regarding their systemic biodistribution after intravenous administration. Methods: Applied to a mouse model of peripheral ischemia, radiolabeled endothelial LEVs were tracked and their in vivo whole-body distribution was quantified by microSPECT/CT imaging. Hindlimb perfusion was followed by LASER Doppler and motility impairment function was evaluated up to day 28 post-ischemia. Results: Early and specific homing of LEVs to ischemic hind limbs was quantified on the day of ischemia and positively correlated with reperfusion intensity at a later stage on day 28 after ischemia, associated with an improved motility function. Conclusions: This concept is a major asset for investigating the biodistribution of LEVs issued from other cell types, including cancer, thus partly contributing to better knowledge and understanding of their fate after injection. (hide) EV-METRIC 67% (94th 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 No extra separation steps Protein markers EV: CD51/ CD63/ CD81/ CD31/ CD146/ caveolin/ ICAM1/ integrin beta 3 non-EV: Albumin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HUVEC EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type JA-30.50 Pelleting: speed (g) 70000 Wash: volume per pellet (ml) 30 Wash: time (min) 90 Wash: Rotor Type JA-30.50 Wash: speed (g) 70000 Other Name other separation method No extra separation steps Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ CD51/ caveolin/ integrin-beta3/ CD81 Not detected contaminants Albumin Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD146/ ICAM1/ CD31 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 434.5 EM EM-type Transmission-EM Image type Close-up Report size (nm) 434

	EV200112	2/2	Homo sapiens	HUVEC	(d)(U)C qEV	Giraud, Romain	2022	67%
Study summary Full title Tracking Radiolabeled Endothelial Microvesicles Predicts Their Therapeutic Efficacy: A Proof-of-Concept Study in Peripheral Ischemia Mouse Model Using SPECT/CT Imaging All authors Romain Giraud, Anaïs Moyon, Stéphanie Simoncini, Anne-Claire Duchez, Vincent Nail, Corinne Chareyre, Ahlem Bouhlel, Laure Balasse, Samantha Fernandez, Loris Vallier, Guillaume Hache, Florence Sabatier, Françoise Dignat-George, Romaric Lacroix, Benjamin Guillet, Philippe Garrigue Journal Pharmaceutics Abstract Microvesicles, so-called endothelial large extracellular vesicles (LEVs), are of great interest as b (show more...)Microvesicles, so-called endothelial large extracellular vesicles (LEVs), are of great interest as biological markers and cell-free biotherapies in cardiovascular and oncologic diseases. However, their therapeutic perspectives remain limited due to the lack of reliable data regarding their systemic biodistribution after intravenous administration. Methods: Applied to a mouse model of peripheral ischemia, radiolabeled endothelial LEVs were tracked and their in vivo whole-body distribution was quantified by microSPECT/CT imaging. Hindlimb perfusion was followed by LASER Doppler and motility impairment function was evaluated up to day 28 post-ischemia. Results: Early and specific homing of LEVs to ischemic hind limbs was quantified on the day of ischemia and positively correlated with reperfusion intensity at a later stage on day 28 after ischemia, associated with an improved motility function. Conclusions: This concept is a major asset for investigating the biodistribution of LEVs issued from other cell types, including cancer, thus partly contributing to better knowledge and understanding of their fate after injection. (hide) EV-METRIC 67% (94th 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 Commercial method Protein markers EV: beta 3 integrin/ CD51/ CD63/ CD81/ CD31/ CD146/ caveolin/ ICAM1 non-EV: Albumin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HUVEC EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type JA-30.50 Pelleting: speed (g) 70000 Wash: volume per pellet (ml) 30 Wash: time (min) 90 Wash: Rotor Type JA-30.50 Wash: speed (g) 70000 Commercial kit qEV 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 CD63/ CD51/ caveolin/ integrin-beta3/ CD81 Not detected contaminants Albumin Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD31/ CD146/ ICAM1 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 434.5 EM EM-type Transmission-EM Image type Close-up Report size (nm) 407

	EV200070	1/2	Homo sapiens	primary CD14+ cells	(d)(U)C	Russell, Ashley	2022	67%
Study summary Full title Cigarette smoke-induced extracellular vesicles from dendritic cells alter T-cell activation and HIV replication All authors Cigarette smoke-induced extracellular vesicles from dendritic cells alter T-cell activation and HIV replication Journal Toxicol Lett. Abstract Despite decreased rates of tobacco smoking in many areas, cigarette smoking remains a major contribu (show more...)Despite decreased rates of tobacco smoking in many areas, cigarette smoking remains a major contributor to many health problems. Cigarette smoking can reduce immune system functioning while concurrently increasing inflammation. Dendritic cells in the lung exposed to cigarette smoke become stimulated and go on to activate T-cells. Extracellular vesicles (EVs) are nano-sized particles released by cells. They participate in intercellular communication by transferring functional proteins and nucleic acids to recipient cells and have been implicated in immune responses. For example, they can display MHC-peptide complexes to activate T-cells. In the current study, we sought to understand the role of cigarette smoke extract (CSE) on dendritic cell-derived EVs and their capacity to activate and differentiate T-cells. Primary human dendritic cells (iDCs) were exposed to CSE and EVs were separated and characterized. We exposed autologous primary CD4 + T-cells to iDC-EVs and observed T helper cell populations skewing towards Th1 and Th17 phenotypes. As HIV + individuals are disproportionately likely to be current smokers, we also examined the effects of iDC-EVs on acutely infected T-cells as well as on a cell model of HIV latency (ACH-2). We found that in most cases, iDC-CSE EVs tended to reduce p24 release from the acutely infected primary T-cells, albeit with great variability. We did not observe large effects of iDC-EVs or direct CSE exposure on p24 release from the ACH-2 cell line. Together, these data suggest that iDC-CSE EVs have the capacity to modulate the immune responses, in part by pushing T-cells towards Th1 and Th17 phenotypes. (hide) EV-METRIC 67% (94th 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 Protein markers EV: CD81/ TSG101/ CD63/ Syntenin non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary CD14+ cells 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: time(min) 90 Pelleting: rotor type AH-629 (36 ml) Pelleting: 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 CD63 Not detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Calnexin/ GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 245.6 nm EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 6.89E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200070	2/2	Homo sapiens	primary CD14+ cells	(d)(U)C	Russell, Ashley	2022	67%
Study summary Full title Cigarette smoke-induced extracellular vesicles from dendritic cells alter T-cell activation and HIV replication All authors Cigarette smoke-induced extracellular vesicles from dendritic cells alter T-cell activation and HIV replication Journal Toxicol Lett. Abstract Despite decreased rates of tobacco smoking in many areas, cigarette smoking remains a major contribu (show more...)Despite decreased rates of tobacco smoking in many areas, cigarette smoking remains a major contributor to many health problems. Cigarette smoking can reduce immune system functioning while concurrently increasing inflammation. Dendritic cells in the lung exposed to cigarette smoke become stimulated and go on to activate T-cells. Extracellular vesicles (EVs) are nano-sized particles released by cells. They participate in intercellular communication by transferring functional proteins and nucleic acids to recipient cells and have been implicated in immune responses. For example, they can display MHC-peptide complexes to activate T-cells. In the current study, we sought to understand the role of cigarette smoke extract (CSE) on dendritic cell-derived EVs and their capacity to activate and differentiate T-cells. Primary human dendritic cells (iDCs) were exposed to CSE and EVs were separated and characterized. We exposed autologous primary CD4 + T-cells to iDC-EVs and observed T helper cell populations skewing towards Th1 and Th17 phenotypes. As HIV + individuals are disproportionately likely to be current smokers, we also examined the effects of iDC-EVs on acutely infected T-cells as well as on a cell model of HIV latency (ACH-2). We found that in most cases, iDC-CSE EVs tended to reduce p24 release from the acutely infected primary T-cells, albeit with great variability. We did not observe large effects of iDC-EVs or direct CSE exposure on p24 release from the ACH-2 cell line. Together, these data suggest that iDC-CSE EVs have the capacity to modulate the immune responses, in part by pushing T-cells towards Th1 and Th17 phenotypes. (hide) EV-METRIC 67% (94th 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 cells exposed to cigarette smoke extract 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 Protein markers EV: TSG101/ CD81/ CD63/ Syntenin non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary CD14+ cells 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: time(min) 90 Pelleting: rotor type AH-629 (36 ml) Pelleting: 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 CD63/ Syntenin/ CD81 Not detected EV-associated proteins TSG101 Detected contaminants Calnexin/ GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 252.45 nm EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 8.51E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220431	2/4	Homo sapiens	Serum	(d)(U)C ExoQuick IgG separation	Desai PP	2022	63%
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 63% (94th 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 Triple negative breast cancer 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 ExoQuick IgG separation Protein markers EV: CD9/ CD81/ TSG101/ Annexin A2/ HSP70 non-EV: Calnexin/ Albumin/ IgG Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Commercial kit ExoQuick Other Name other separation method ExoQuick Selected surface protein(s) Annexin A2 Other Name other separation method IgG separation 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/ CD81/ TSG101/ Annexin A2 Not detected EV-associated proteins HSP70 Detected contaminants Albumin/ IgG Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 77-106 EV concentration Yes Particle yield particles per milliliter of starting sample: 3.89E+10 EM EM-type Cryo-EM Image type Close-up Report size (nm) 20-80

	EV220389	1/2	Homo sapiens	Blood plasma	ExoQuick	Murugesan S	2022	63%
Study summary Full title Extracellular Vesicles From Women With Severe Preeclampsia Impair Vascular Endothelial Function. All authors Murugesan S, Hussey H, Saravanakumar L, Sinkey RG, Sturdivant AB, Powell MF, Berkowitz DE Journal Anesth Analg Abstract Preeclampsia (PE) manifesting as hypertension and organ injury is mediated by vascular dysfunction. (show more...)Preeclampsia (PE) manifesting as hypertension and organ injury is mediated by vascular dysfunction. In biological fluids, extracellular vesicles (EVs) containing microRNA (miRNA), protein, and other cargo released from the placenta may serve as carriers to propagate injury, altering the functional phenotype of endothelial cells. PE has been consistently correlated with increased levels of placenta-derived EVs (pEVs) in maternal circulation. However, whether pEVs impaired endothelial cell function remains to be determined. In this study, we hypothesize that pEVs from pregnant women with severe PE (sPE) impair endothelial function through altered cell signaling. (hide) EV-METRIC 63% (91st 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 Blood plasma Sample origin Healthy pregnant 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 ExoQuick Protein markers EV: CD63/ Flotillin1/ PLAP/ TSG101 non-EV: Apolipoprotein A1/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method BCA 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 CD63/ Flotillin1 Not detected contaminants Apolipoprotein A1/ GM130 ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ PLAP Other 1 Confocal microscopy Detected EV-associated proteins TSG101 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 101.3 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.28E+06 EM EM-type Transmission-EM Image type Close-up Report size (nm) 30-150

	EV220389	2/2	Homo sapiens	Blood plasma	ExoQuick	Murugesan S	2022	63%
Study summary Full title Extracellular Vesicles From Women With Severe Preeclampsia Impair Vascular Endothelial Function. All authors Murugesan S, Hussey H, Saravanakumar L, Sinkey RG, Sturdivant AB, Powell MF, Berkowitz DE Journal Anesth Analg Abstract Preeclampsia (PE) manifesting as hypertension and organ injury is mediated by vascular dysfunction. (show more...)Preeclampsia (PE) manifesting as hypertension and organ injury is mediated by vascular dysfunction. In biological fluids, extracellular vesicles (EVs) containing microRNA (miRNA), protein, and other cargo released from the placenta may serve as carriers to propagate injury, altering the functional phenotype of endothelial cells. PE has been consistently correlated with increased levels of placenta-derived EVs (pEVs) in maternal circulation. However, whether pEVs impaired endothelial cell function remains to be determined. In this study, we hypothesize that pEVs from pregnant women with severe PE (sPE) impair endothelial function through altered cell signaling. (hide) EV-METRIC 63% (91st 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 Blood plasma Sample origin Severe pre-eclampsia 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 ExoQuick Protein markers EV: CD63/ Flotillin1/ PLAP/ TSG101 non-EV: Apolipoprotein A1/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method BCA 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 CD63/ Flotillin1 Not detected contaminants Apolipoprotein A1/ GM130 ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ PLAP Other 1 Confocal microscopy Detected EV-associated proteins TSG101 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 108.8 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.63E+06 EM EM-type Transmission-EM Image type Close-up Report size (nm) 30-150

	EV220091	2/8	Homo sapiens	HeLa	UF SEC (non-commercial)	Visan, Kekoolani	2022	63%
Study summary Full title Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles All authors Kekoolani S. Visan, Richard J. Lobb, Sunyoung Ham, Luize G. Lima, Carlos Palma, Chai Pei Zhi Edna, Li-Ying Wu, Harsha Gowda, Keshava K. Datta, Gunter Hartel, Carlos Salomon, Andreas Möller Journal J Extracell Vesicles Abstract Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, progno (show more...)Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum-containing media. The majority of researchers use serum-containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non-sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA-MB-231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano-flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large-scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations. (hide) EV-METRIC 63% (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 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 Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ Flotillin-1/ HSP70/ TSG101 non-EV: Calnexin/ Albumin Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method Ultra filtration Cut-off size (kDa) 300 Membrane type Polyethersulfone (PES) Size-exclusion chromatography Used for validation? Yes Total column volume (mL) 10 Sample volume/column (mL) 0.5 Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD9/ Flotillin-1/ HSP70/ TSG101 Detected contaminants Albumin Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 133 EV concentration Yes Particle yield Total Particles: 4.45E+10 TRPS Report type Modus Reported size (nm) 98.33333333 EV concentration Yes Particle yield Total Particles: 9.01E+08 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220003	2/2	Homo sapiens	Serum	DG SEC (non-commercial)	Karimi, Nasibeh	2022	63%
Study summary Full title Tetraspanins distinguish separate extracellular vesicle subpopulations in human serum and plasma - Contributions of platelet extracellular vesicles in plasma samples All authors Nasibeh Karimi, Razieh Dalirfardouei, Tomás Dias, Jan Lötvall, Cecilia Lässer Journal J Extracell Vesicles Abstract Background: The ability to isolate extracellular vesicles (EVs) from blood is vital in the developme (show more...)Background: The ability to isolate extracellular vesicles (EVs) from blood is vital in the development of EVs as disease biomarkers. Both serum and plasma can be used, but few studies have compared these sources in terms of the type of EVs that are obtained. The aim of this study was to determine the presence of different subpopulations of EVs in plasma and serum. Method: Blood was collected from healthy subjects, and plasma and serum were isolated in parallel. ACD or EDTA tubes were used for the collection of plasma, while serum was obtained in clot activator tubes. EVs were isolated utilising a combination of density cushion and SEC, a combination of density cushion and gradient or by a bead antibody capturing system (anti-CD63, anti-CD9 and anti-CD81 beads). The subpopulations of EVs were analysed by NTA, Western blot, SP-IRIS, conventional and nano flow cytometry, magnetic bead ELISA and mass spectrometry. Additionally, different isolation protocols for plasma were compared to determine the contribution of residual platelets in the analysis. Results: This study shows that a higher number of CD9+ EVs were present in EDTA-plasma compared to ACD-plasma and to serum, and the presence of CD41a on these EVs suggests that they were released from platelets. Furthermore, only a very small number of EVs in blood were double-positive for CD63 and CD81. The CD63+ EVs were enriched in serum, while CD81+ vesicles were the rarest subpopulation in both plasma and serum. Additionally, EDTA-plasma contained more residual platelets than ACD-plasma and serum, and two centrifugation steps were crucial to reduce the number of platelets in plasma prior to EV isolation. Conclusion: These results show that human blood contains multiple subpopulations of EVs that carry different tetraspanins. Blood sampling methods, including the use of anti-coagulants and choice of centrifugation protocols, can affect EV analyses and should always be reported in detail. (hide) EV-METRIC 63% (94th 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 Density gradient Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD63/ CD81/ Flotillin-1/ CD41a/ P-selectin non-EV: None Proteomics no EV density (g/ml) 1.06-1.16 Show all info Study aim Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Serum Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 50% Total gradient volume, incl. sample (mL) 12 ml Sample volume (mL) 6 ml Orientation Top-down Rotor type SW 41 Ti Speed (g) 178,000 Duration (min) 180 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) 26.7 Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81 Not detected EV-associated proteins Flotillin-1/ CD41a/ P-selectin Detected EV-associated proteins CD9/ CD63/ CD81/ CD41a Detected EV-associated proteins CD9/ CD63/ CD81/ CD41a Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield as total number of particles in pooled SEC fractions from 6 mL of sample: 2.56e+9 Report type Size range/distribution Report size 50-70 EV-concentration No

	EV210210	1/1	Homo sapiens	Expi293F	(d)(U)C Tangential flow filtration qEV10 Filtration UF	Driedonks T	2022	63%
Study summary Full title Pharmacokinetics and biodistribution of extracellular vesicles administered intravenously and intranasally to . All authors Driedonks T, Jiang L, Carlson B, Han Z, Liu G, Queen SE, Shirk EN, Gololobova O, Liao Z, Nyberg LH, Lima G, Paniushkina L, Garcia-Contreras M, Schonvisky K, Castell N, Stover M, Guerrero-Martin S, Richardson R, Smith B, Machairaki V, Lai CP, Izzi JM, Hutchinson EK, Pate KAM, Witwer KW Journal J Extracell Biol Abstract Extracellular vesicles (EVs) have potential in disease treatment since they can be loaded with thera (show more...)Extracellular vesicles (EVs) have potential in disease treatment since they can be loaded with therapeutic molecules and engineered for retention by specific tissues. However, questions remain on optimal dosing, administration, and pharmacokinetics. Previous studies have addressed biodistribution and pharmacokinetics in rodents, but little evidence is available for larger animals. Here, we investigated the pharmacokinetics and biodistribution of Expi293F-derived EVs labelled with a highly sensitive nanoluciferase reporter (palmGRET) in a non-human primate model (Macaca nemestrina), comparing intravenous (IV) and intranasal (IN) administration over a 125-fold dose range. We report that EVs administered IV had longer circulation times in plasma than previously reported in mice and were detectable in cerebrospinal fluid (CSF) after 30-60 minutes. EV association with PBMCs, especially B-cells, was observed as early as one minute post-administration. EVs were detected in liver and spleen within one hour of IV administration. However, IN delivery was minimal, suggesting that pretreatment approaches may be needed in large animals. Furthermore, EV circulation times strongly decreased after repeated IV administration, possibly due to immune responses and with clear implications for xenogeneic EV-based therapeutics. We hope that our findings from this baseline study in macaques will help to inform future research and therapeutic development of EVs. (hide) EV-METRIC 63% (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 Cell culture supernatant Sample origin pLenti-palmGRET (Addgene #158221) transient expression 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 Tangential flow filtration Commercial method Filtration Ultrafiltration Protein markers EV: CD9/ CD63/ CD81/ TSG101/ CD29/ CD146/ CD326/ CD44/ ROR1/ CD24 non-EV: Calnexin Proteomics no Show all info Study aim Biodistribution/pharmacokinetics/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F Cell count 3.30E+09 Separation Method Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Commercial kit qEV10 Other Name other separation method Tangential flow filtration Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ TSG101/ CD47 Not detected contaminants Calnexin Other 1 SP-IRIS (Exoview) Detected EV-associated proteins CD63/ CD81 Other 2 MACSPlex Detected EV-associated proteins CD9/ CD63/ CD81/ CD29/ CD146/ CD326/ CD44/ ROR1/ CD24 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 122.6 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type Amnis ImageStream MKII Hardware adjustment EM EM-type Transmission-EM Image type Wide-field Extra information Size-exclusion chromatography details: Izon qEV10, first 20 ml after the void fraction were considered EV-enriched fractions/ Tangential Flow Filtration: Sartorius Vivaflow 50R cassettes, 100 kD

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Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

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Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data