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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV170028	1/1	Homo sapiens	Synovial fluid	DG (d)(U)C SEC	Foers AD	2017	100%
Study summary Full title Enrichment of extracellular vesicles from human synovial fluid using size exclusion chromatography. All authors Foers AD, Chatfield S, Dagley LF, Scicluna BJ, Webb AI, Cheng L, Hill AF, Wicks IP, Pang KC. Journal J Extracell Vesicles Abstract As a complex biological fluid, human synovial fluid (SF) presents challenges for extracellular vesic (show more...)As a complex biological fluid, human synovial fluid (SF) presents challenges for extracellular vesicle (EV) enrichment using standard methods. In this study of human SF, a size exclusion chromatography (SEC)-based method of EV enrichment is shown to deplete contaminants that remain after standard ultracentrifugation-based enrichment methods. Specifically, considerable levels of serum albumin, the high-density lipoprotein marker, apolipoprotein A-I, fibronectin and other extracellular proteins and debris are present in EVs prepared by differential ultracentrifugation. While the addition of a sucrose density gradient purification step improved purification quality, some contamination remained. In contrast, using a SEC-based approach, SF EVs were efficiently separated from serum albumin, apolipoprotein A-I and additional contaminating proteins that co-purified with high-speed centrifugation. Finally, using high-resolution mass spectrometry analysis, we found that residual contaminants which remain after SEC, such as fibronectin and other extracellular proteins, can be successfully depleted by proteinase K. Taken together, our results highlight the limitations of ultracentrifugation-based methods of EV isolation from complex biological fluids and suggest that SEC can be used to obtain higher purity EV samples. In this way, SEC-based methods are likely to be useful for identifying EV-enriched components and improving understanding of EV function in disease. (hide) EV-METRIC 100% (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 Synovial fluid Sample origin Arthritis 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 DG (d)(U)C SEC Adj. k-factor 156.9 (pelleting) / 156.9 (washing) Protein markers EV: TSG101/ Rab27b/ Annexin1/ Syntenin/ HSP70/ Flotillin-1 non-EV: Fibronectin/ Albumin/ ApoA1 Proteomics yes Show all info Study aim New methodological development, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Synovial fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Wash: time (min) 90 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 156.9 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 0.25 Highest density fraction 2 Sample volume (mL) 0.65 Orientation Top-down (sample migrates downwards) Rotor type SW 40 Ti Speed (g) 202000 Duration (min) 1100 Fraction volume (mL) 1.05 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: duration (min) 90 Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 122.2 Size-exclusion chromatography Total column volume (mL) 320 Sample volume/column (mL) 13 Resin type Sephacryl S-500 HR Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin-1, HSP70, TSG101, Syntenin, Rab27b, Annexin1 Not detected contaminants Albumin, ApoA1 Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) x EV concentration Yes Particle yield x EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV170008	1/3	Homo sapiens	primary monocyte derived dendritic cell	DG (d)(U)C	Mercedes Tkach	2017	100%
Study summary Full title Qualitative differences in T-cell activation by dendritic cell-derived extracellular vesicle subtypes. All authors Tkach M, Kowal J, Zucchetti AE, Enserink L, Jouve M, Lankar D, Saitakis M, Martin-Jaular L, Théry C Journal EMBO J Abstract Exosomes, nano-sized secreted extracellular vesicles (EVs), are actively studied for their diagnosti (show more...)Exosomes, nano-sized secreted extracellular vesicles (EVs), are actively studied for their diagnostic and therapeutic potential. In particular, exosomes secreted by dendritic cells (DCs) have been shown to carry MHC-peptide complexes allowing efficient activation of T lymphocytes, thus displaying potential as promoters of adaptive immune responses. DCs also secrete other types of EVs of different size, subcellular origin and protein composition, whose immune capacities have not been yet compared to those of exosomes. Here, we show that large EVs (lEVs) released by human DCs are as efficient as small EVs (sEVs), including exosomes, to induce CD4+ T-cell activation in vitro When released by immature DCs, however, lEVs and sEVs differ in their capacity to orient T helper (Th) cell responses, the former favouring secretion of Th2 cytokines, whereas the latter promote Th1 cytokine secretion (IFN-γ). Upon DC maturation, however, these functional differences are abolished, and all EVs become able to induce IFN-γ. Our results highlight the need to comprehensively compare the functionalities of EV subtypes in all patho/physiological systems where exosomes are claimed to perform critical roles. (hide) EV-METRIC 100% (99th 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 DG (d)(U)C Protein markers EV: CD63/ MHC2/ CD9/ CD9,MHC2 non-EV: None Proteomics no Show all info Study aim EV functional activity Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary monocyte derived dendritic cell EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) NA Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 20 Pelleting: rotor type Swinging bucket Pelleting: speed (g) 2000 Wash: time (min) 20 Wash: Rotor Type Eppendorf 5810R cf; swinging bucket Wash: speed (g) 2000 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 0.1 Highest density fraction 0.3 Sample volume (mL) 1.2 Orientation Bottom-up (sample migrates upwards) Rotor type SW 55 Ti Speed (g) 350000 Duration (min) 60 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 2.5 Pelleting: duration (min) 30 Pelleting: rotor type TLA-110 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 65.69 EV-subtype Distinction between multiple subtypes Centrifugation steps: 2K, 10K, 100K PMID previous EV protein analysis 26858453 Protein Concentration Method microBCA Protein Yield (µg) 2.9+-0.3 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9,MHC2 Flow cytometry Type of Flow cytometry MACSQuant Miltenyi Calibration bead size 0.1-0.3,0.4-0.6,0.7-0.9,1.0-1.9 Antibody details provided? No Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis 26858453 Extra particle analysis NTA Report type Size range/distribution EV concentration Yes Particle yield 6.52E10+-2.03E10 particles/million cells EM EM-type Transmission-EM/ Scanning-EM EM protein MHC2 Image type Close-up, Wide-field

	EV170008	2/3	Homo sapiens	primary monocyte derived dendritic cell	DG (d)(U)C	Mercedes Tkach	2017	100%
Study summary Full title Qualitative differences in T-cell activation by dendritic cell-derived extracellular vesicle subtypes. All authors Tkach M, Kowal J, Zucchetti AE, Enserink L, Jouve M, Lankar D, Saitakis M, Martin-Jaular L, Théry C Journal EMBO J Abstract Exosomes, nano-sized secreted extracellular vesicles (EVs), are actively studied for their diagnosti (show more...)Exosomes, nano-sized secreted extracellular vesicles (EVs), are actively studied for their diagnostic and therapeutic potential. In particular, exosomes secreted by dendritic cells (DCs) have been shown to carry MHC-peptide complexes allowing efficient activation of T lymphocytes, thus displaying potential as promoters of adaptive immune responses. DCs also secrete other types of EVs of different size, subcellular origin and protein composition, whose immune capacities have not been yet compared to those of exosomes. Here, we show that large EVs (lEVs) released by human DCs are as efficient as small EVs (sEVs), including exosomes, to induce CD4+ T-cell activation in vitro When released by immature DCs, however, lEVs and sEVs differ in their capacity to orient T helper (Th) cell responses, the former favouring secretion of Th2 cytokines, whereas the latter promote Th1 cytokine secretion (IFN-γ). Upon DC maturation, however, these functional differences are abolished, and all EVs become able to induce IFN-γ. Our results highlight the need to comprehensively compare the functionalities of EV subtypes in all patho/physiological systems where exosomes are claimed to perform critical roles. (hide) EV-METRIC 100% (99th 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 DG (d)(U)C Adj. k-factor 2097 (pelleting) / 2097 (washing) Protein markers EV: CD63/ MHC2/ CD9/ CD9,MHC2 non-EV: None Proteomics no Show all info Study aim EV functional activity Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary monocyte derived dendritic cell EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) NA 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: time(min) 40 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 10000 Pelleting: adjusted k-factor 2097. Wash: time (min) 40 Wash: Rotor Type Type 45 Ti Wash: speed (g) 10000 Wash: adjusted k-factor 2097. Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 0.1 Highest density fraction 0.3 Sample volume (mL) 1.2 Orientation Bottom-up (sample migrates upwards) Rotor type SW 55 Ti Speed (g) 350000 Duration (min) 60 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 2.5 Pelleting: duration (min) 30 Pelleting: rotor type TLA-110 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 65.69 EV-subtype Distinction between multiple subtypes Centrifugation steps: 2K, 10K, 100K PMID previous EV protein analysis 26858453 Protein Concentration Method microBCA Protein Yield (µg) 1.7+-0.3 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9,MHC2 Flow cytometry Type of Flow cytometry MACSQuant Miltenyi Calibration bead size 0.1-0.3,0.4-0.6,0.7-0.9,1.0-1.9 Antibody details provided? No Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis 26858453 Extra particle analysis NTA Report type Size range/distribution EV concentration Yes Particle yield 4.95E10+-3.81E10 particles/million cells EM EM-type Transmission-EM/ Scanning-EM EM protein MHC2 Image type Close-up, Wide-field

	EV170008	3/3	Homo sapiens	primary monocyte derived dendritic cell	DG (d)(U)C	Mercedes Tkach	2017	100%
Study summary Full title Qualitative differences in T-cell activation by dendritic cell-derived extracellular vesicle subtypes. All authors Tkach M, Kowal J, Zucchetti AE, Enserink L, Jouve M, Lankar D, Saitakis M, Martin-Jaular L, Théry C Journal EMBO J Abstract Exosomes, nano-sized secreted extracellular vesicles (EVs), are actively studied for their diagnosti (show more...)Exosomes, nano-sized secreted extracellular vesicles (EVs), are actively studied for their diagnostic and therapeutic potential. In particular, exosomes secreted by dendritic cells (DCs) have been shown to carry MHC-peptide complexes allowing efficient activation of T lymphocytes, thus displaying potential as promoters of adaptive immune responses. DCs also secrete other types of EVs of different size, subcellular origin and protein composition, whose immune capacities have not been yet compared to those of exosomes. Here, we show that large EVs (lEVs) released by human DCs are as efficient as small EVs (sEVs), including exosomes, to induce CD4+ T-cell activation in vitro When released by immature DCs, however, lEVs and sEVs differ in their capacity to orient T helper (Th) cell responses, the former favouring secretion of Th2 cytokines, whereas the latter promote Th1 cytokine secretion (IFN-γ). Upon DC maturation, however, these functional differences are abolished, and all EVs become able to induce IFN-γ. Our results highlight the need to comprehensively compare the functionalities of EV subtypes in all patho/physiological systems where exosomes are claimed to perform critical roles. (hide) EV-METRIC 100% (99th 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 DG (d)(U)C Adj. k-factor 209.7 (pelleting) / 209.7 (washing) Protein markers EV: CD63/ MHC2/ CD9/ CD9,MHC2 non-EV: None Proteomics no Show all info Study aim EV functional activity Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary monocyte derived dendritic cell EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) NA 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) 100000 Pelleting: adjusted k-factor 209.7 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 209.7 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 0.1 Highest density fraction 0.3 Sample volume (mL) 1.2 Orientation Bottom-up (sample migrates upwards) Rotor type SW 55 Ti Speed (g) 350000 Duration (min) 60 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 2.5 Pelleting: duration (min) 30 Pelleting: rotor type TLA-110 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 65.69 EV-subtype Distinction between multiple subtypes Centrifugation steps: 2K, 10K, 100K PMID previous EV protein analysis 26858453 Protein Concentration Method microBCA Protein Yield (µg) 1.1+-0.2 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9,MHC2 Flow cytometry Type of Flow cytometry MACSQuant Miltenyi Calibration bead size 0.1-0.3,0.4-0.6,0.7-0.9,1.0-1.9 Antibody details provided? No Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis 26858453 Extra particle analysis NTA Report type Size range/distribution EV concentration Yes Particle yield 3.76E10+-1.41E10 particles/million cells EM EM-type Transmission-EM/ Scanning-EM EM protein MHC2 Image type Close-up, Wide-field

	EV170001	1/1	Homo sapiens	MCF7 Rab27b-GFP	DG (d)(U)C Filtration UF	Vergauwen, Glenn	2017	100%
Study summary Full title Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research. All authors Vergauwen G, Dhondt B, Van Deun J, De Smedt E, Berx G, Timmerman E, Gevaert K, Miinalainen I, Cocquyt V, Braems G, Van den Broecke R, Denys H, De Wever O, Hendrix A. Journal Sci Rep Abstract Identification and validation of extracellular vesicle (EV)-associated biomarkers requires robust is (show more...)Identification and validation of extracellular vesicle (EV)-associated biomarkers requires robust isolation and characterization protocols. We assessed the impact of some commonly implemented pre-analytical, analytical and post-analytical variables in EV research. Centrifugal filters with different membrane types and pore sizes are used to reduce large volume biofluids prior to EV isolation or to concentrate EVs. We compared five commonly reported filters for their efficiency when using plasma, urine and EV-spiked PBS. Regenerated cellulose membranes with pore size of 10 kDa recovered EVs the most efficient. Less than 40% recovery was achieved with other filters. Next, we analyzed the effect of the type of protein assays to measure EV protein in colorimetric and fluorometric kits. The fluorometric assay Qubit measured low concentration EV and BSA samples the most accurately with the lowest variation among technical and biological replicates. Lastly, we quantified Optiprep remnants in EV samples from density gradient ultracentrifugation and demonstrate that size-exclusion chromatography efficiently removes Optiprep from EVs. In conclusion, choice of centrifugal filters and protein assays confound EV analysis and should be carefully considered to increase efficiency towards biomarker discovery. SEC-based removal of Optiprep remnants from EVs can be considered for downstream applications. (hide) EV-METRIC 100% (99th 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 DG (d)(U)C Filtration UF Protein markers EV: Alix/ CD81/ TSG101 non-EV: PMP70/ Argonaute-2 Proteomics no EV density (g/ml) 1.094 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF7 Rab27b-GFP EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No 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) 16.5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,…) Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD81/ TSG101 Not detected contaminants Argonaute-2/ PMP70 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Characterization: Particle analysis NTA Report type Modus Reported size (nm) 113 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV170046	1/2	Sus scrofa	Primary retinal pigmented epithelium cells	DG (d)(U)C UF	Klingeborn M	2017	88%
Study summary Full title Directional Exosome Proteomes Reflect Polarity-Specific Functions in Retinal Pigmented Epithelium Monolayers. All authors Klingeborn M, Dismuke WM, Skiba NP, Kelly U, Stamer WD, Bowes Rickman C Journal Sci Rep Abstract The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier in the eye and its pola (show more...)The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier in the eye and its polarity is responsible for directional secretion and uptake of proteins, lipoprotein particles and extracellular vesicles (EVs). Such a secretional division dictates directed interactions between the systemic circulation (basolateral) and the retina (apical). Our goal is to define the polarized proteomes and physical characteristics of EVs released from the RPE. Primary cultures of porcine RPE cells were differentiated into polarized RPE monolayers on permeable supports. EVs were isolated from media bathing either apical or basolateral RPE surfaces, and two subpopulations of small EVs including exosomes, and dense EVs, were purified and processed for proteomic profiling. In parallel, EV size distribution and concentration were determined. Using protein correlation profiling mass spectrometry, a total of 631 proteins were identified in exosome preparations, 299 of which were uniquely released apically, and 94 uniquely released basolaterally. Selected proteins were validated by Western blot. The proteomes of these exosome and dense EVs preparations suggest that epithelial polarity impacts directional release. These data serve as a foundation for comparative studies aimed at elucidating the role of exosomes in the molecular pathophysiology of retinal diseases and help identify potential therapeutic targets and biomarkers. (hide) EV-METRIC 88% (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 Cell culture supernatant Sample origin Basolateral side of polarized layer 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 DG (d)(U)C UF Adj. k-factor 253.9 (pelleting) / 253.9 (washing) Protein markers EV: TSG101/ Syntenin-1 non-EV: Calreticulin Proteomics yes Show all info Study aim New methodological development, Identification of content (omics approaches) Sample Species Sus scrofa Sample Type Cell culture supernatant EV-producing cells Primary retinal pigmented epithelium cells EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Wash: time (min) 90 Wash: Rotor Type SW 28 Wash: speed (g) 100000 Wash: adjusted k-factor 253.9 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 1 Orientation Bottom-up (sample migrates upwards) Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: duration (min) 90 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 255.8 Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Characterization: Protein analysis Protein Concentration Method Pierce 660nm Protein Assay Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins TSG101, Syntenin-1 Not detected contaminants Calreticulin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 132.2±13.1 EM EM-type Transmission-EM Image type Wide-field

	EV170046	2/2	Sus scrofa	Primary retinal pigmented epithelium cells	DG (d)(U)C UF	Klingeborn M	2017	88%
Study summary Full title Directional Exosome Proteomes Reflect Polarity-Specific Functions in Retinal Pigmented Epithelium Monolayers. All authors Klingeborn M, Dismuke WM, Skiba NP, Kelly U, Stamer WD, Bowes Rickman C Journal Sci Rep Abstract The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier in the eye and its pola (show more...)The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier in the eye and its polarity is responsible for directional secretion and uptake of proteins, lipoprotein particles and extracellular vesicles (EVs). Such a secretional division dictates directed interactions between the systemic circulation (basolateral) and the retina (apical). Our goal is to define the polarized proteomes and physical characteristics of EVs released from the RPE. Primary cultures of porcine RPE cells were differentiated into polarized RPE monolayers on permeable supports. EVs were isolated from media bathing either apical or basolateral RPE surfaces, and two subpopulations of small EVs including exosomes, and dense EVs, were purified and processed for proteomic profiling. In parallel, EV size distribution and concentration were determined. Using protein correlation profiling mass spectrometry, a total of 631 proteins were identified in exosome preparations, 299 of which were uniquely released apically, and 94 uniquely released basolaterally. Selected proteins were validated by Western blot. The proteomes of these exosome and dense EVs preparations suggest that epithelial polarity impacts directional release. These data serve as a foundation for comparative studies aimed at elucidating the role of exosomes in the molecular pathophysiology of retinal diseases and help identify potential therapeutic targets and biomarkers. (hide) EV-METRIC 88% (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 Cell culture supernatant Sample origin Apical side of polarized layer 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 DG (d)(U)C UF Adj. k-factor 253.9 (pelleting) / 253.9 (washing) Protein markers EV: TSG101/ Syntenin-1 non-EV: Calreticulin Proteomics yes Show all info Study aim New methodological development, Identification of content (omics approaches) Sample Species Sus scrofa Sample Type Cell culture supernatant EV-producing cells Primary retinal pigmented epithelium cells EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Wash: time (min) 90 Wash: Rotor Type SW 28 Wash: speed (g) 100000 Wash: adjusted k-factor 253.9 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 1 Orientation Bottom-up (sample migrates upwards) Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: duration (min) 90 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 255.8 Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Characterization: Protein analysis Protein Concentration Method Pierce 660nm Protein Assay Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins TSG101, Syntenin-1 Not detected contaminants Calreticulin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 118.6±9.9 EM EM-type Transmission-EM Image type Close-up

	EV170016	1/2	Mus musculus	adipose tissue-derived macrophages	DG (d)(U)C Filtration	Ying, Wei	2017	88%
Study summary Full title Adipose Tissue Macrophage-Derived Exosomal miRNAs Can Modulate In Vivo and In Vitro Insulin Sensitivity. All authors Ying W, Riopel M, Bandyopadhyay G, Dong Y, Birmingham A, Seo JB, Ofrecio JM, Wollam J, Hernandez-Carretero A, Fu W, Li P, Olefsky JM Journal Cell Abstract MiRNAs are regulatory molecules that can be packaged into exosomes and secreted from cells. Here, we (show more...)MiRNAs are regulatory molecules that can be packaged into exosomes and secreted from cells. Here, we show that adipose tissue macrophages (ATMs) in obese mice secrete miRNA-containing exosomes (Exos), which cause glucose intolerance and insulin resistance when administered to lean mice. Conversely, ATM Exos obtained from lean mice improve glucose tolerance and insulin sensitivity when administered to obese recipients. miR-155 is one of the miRNAs overexpressed in obese ATM Exos, and earlier studies have shown that PPARγ is a miR-155 target. Our results show that miR-155KO animals are insulin sensitive and glucose tolerant compared to controls. Furthermore, transplantation of WT bone marrow into miR-155KO mice mitigated this phenotype. Taken together, these studies show that ATMs secrete exosomes containing miRNA cargo. These miRNAs can be transferred to insulin target cell types through mechanisms of paracrine or endocrine regulation with robust effects on cellular insulin action, in vivo insulin sensitivity, and overall glucose homeostasis. (hide) EV-METRIC 88% (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 Cell culture supernatant Sample origin Obesitas 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 DG (d)(U)C Filtration Adj. k-factor 256 (pelleting) / 256 (washing) Protein markers EV: TSG101/ HSP70/ Syntenin1/ CD63/ CD9 non-EV: Grp94 Proteomics no Show all info Study aim Function, Mechanism of uptake/transfer Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells adipose tissue-derived macrophages EV-harvesting Medium EV-depleted serum Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 240-360 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 256.0 Wash: time (min) 20 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 256.0 Density gradient Only used for validation of main results Yes Density medium 156.9 Type Discontinuous Lowest density fraction 0.1 Highest density fraction 0.3 Sample volume (mL) 0.3 Orientation Bottom-up (sample migrates upwards) Rotor type Type 70 Ti Speed (g) 350000 Duration (min) 60 Fraction volume (mL) 2.4 Fraction processing Centrifugation Pelleting: volume per fraction 2 Pelleting: duration (min) 90 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Pelleting-wash: volume per pellet (mL) 24 Pelleting-wash: duration (min) 30 Pelleting-wash: rotor type 156.9 Pelleting-wash: speed (g) Type 70 Ti Pelleting-wash: adjusted k-factor 156.9 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes 1.13-1.15 g/ml Used subtypes Yes Characterization: Protein analysis Protein Concentration Method DC protein assay Protein Yield (µg) 9-May Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63, HSP70, TSG101, Syntenin1 Not detected contaminants Grp94 Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-150 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 50-200 nm Extra information Full UC and density gradient protocols not in original article

	EV170004	1/1	Homo sapiens	Adipose tissue	DG (d)(U)C Filtration	Jeurissen S	2017	88%
Study summary Full title The isolation of morphologically intact and biologically active extracellular vesicles from the secretome of cancer-associated adipose tissue. All authors Jeurissen S, Vergauwen G, Van Deun J, Lapeire L, Depoorter V, Miinalainen I, Sormunen R, Van den Broecke R, Braems G, Cocquyt V, Denys H, Hendrix A Journal Cell Adh Migr Abstract Breast cancer cells closely interact with different cell types of the surrounding adipose tissue to (show more...)Breast cancer cells closely interact with different cell types of the surrounding adipose tissue to favor invasive growth and metastasis. Extracellular vesicles (EVs) are nanometer-sized vesicles secreted by different cell types that shuttle proteins and nucleic acids to establish cell-cell communication. To study the role of EVs released by cancer-associated adipose tissue in breast cancer progression and metastasis a standardized EV isolation protocol that obtains pure EVs and maintains their functional characteristics is required. We implemented differential ultracentrifugation as a pre-enrichment step followed by OptiPrep density gradient centrifugation (dUC-ODG) to isolate EVs from the conditioned medium of cancer-associated adipose tissue. A combination of immune-electron microscopy, nanoparticle tracking analysis (NTA) and Western blot analysis identified EVs that are enriched in flotillin-1, CD9 and CD63, and sized between 20 and 200 nm with a density of 1.076-1.125 g/ml. The lack of protein aggregates and cell organelle proteins confirmed the purity of the EV preparations. Next, we evaluated whether dUC-ODG isolated EVs are functionally active. ZR75.1 breast cancer cells treated with cancer-associated adipose tissue-secreted EVs from breast cancer patients showed an increased phosphorylation of CREB. MCF-7 breast cancer cells treated with adipose tissue-derived EVs exhibited a stronger propensity to form cellular aggregates. In conclusion, dUC-ODG purifies EVs from conditioned medium of cancer-associated adipose tissue, and these EVs are morphologically intact and biologically active. (hide) EV-METRIC 88% (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 Adipose tissue Sample origin breast cancer 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 DG (d)(U)C Filtration Adj. k-factor 138.6 (pelleting) / 138.6 (washing) Protein markers EV: CD63/ HSP70/ FABP4/ Flotillin-1/ CD9 non-EV: Prohibitin/ GM130/ Calreticulin Proteomics no Show all info Study aim Function, Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Adipose 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: time(min) 120 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 138.6 Wash: time (min) 120 Wash: Rotor Type SW 55 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 138.6 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 1 Orientation Top-down (sample migrates downwards) Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 297.9 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD9, Flotillin-1, HSP70, FABP4 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 116 EV concentration Yes Particle yield 2.2 10E09 EM EM-type Immune-EM EM protein CD63 Image type Close-up, Wide-field Report size (nm) 20-200

	EV210202	1/7	Mus musculus	3T3-L1	DG (d)(U)C	Durcin, Maëva	2017	78%
Study summary Full title Characterisation of adipocyte-derived extracellular vesicle subtypes identifies distinct protein and lipid signatures for large and small extracellular vesicles All authors Maëva Durcin, Audrey Fleury, Emiliane Taillebois, Grégory Hilairet, Zuzana Krupova, Céline Henry, Sandrine Truchet, Martin Trötzmüller, Harald Köfeler, Guillaume Mabilleau, Olivier Hue, Ramaroson Andriantsitohaina, Patrice Martin, Soazig Le Lay Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells (show more...)Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Large extracellular vesicles 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 DG (d)(U)C Protein markers EV: None non-EV: None Proteomics yes EV density (g/ml) 1.14-1.24 Show all info Study aim Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells 3T3-L1 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 Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type MLA-50 Pelleting: speed (g) 13000 Wash: volume per pellet (ml) Not specified Wash: time (min) 60 Wash: Rotor Type MLA-50 Wash: speed (g) 13000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 31 Lowest density fraction 0.4M Highest density fraction 2M Total gradient volume, incl. sample (mL) 12000 Sample volume (mL) 500 Orientation Top-down Rotor type Not specified Speed (g) 200000 Duration (min) 1080 Fraction volume (mL) 1000 Fraction processing Centrifugation Pelleting: volume per fraction 6000 Pelleting: duration (min) 70 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ Mfge8/ caveolin-1/ b-actin/ Flotillin2/ TSG101/ Alix Not detected EV-associated proteins CD81 Flow cytometry Type of Flow cytometry MACSQuant flow cytometer Calibration bead size 4µm Antibody details provided? No Detected EV-associated proteins Annexin V Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 160 EV concentration Yes Particle yield EV secreted per adipocyte;Yes, other: 11 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-100

	EV210202	2/7	Mus musculus	3T3-L1	DG (d)(U)C	Durcin, Maëva	2017	78%
Study summary Full title Characterisation of adipocyte-derived extracellular vesicle subtypes identifies distinct protein and lipid signatures for large and small extracellular vesicles All authors Maëva Durcin, Audrey Fleury, Emiliane Taillebois, Grégory Hilairet, Zuzana Krupova, Céline Henry, Sandrine Truchet, Martin Trötzmüller, Harald Köfeler, Guillaume Mabilleau, Olivier Hue, Ramaroson Andriantsitohaina, Patrice Martin, Soazig Le Lay Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells (show more...)Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues. (hide) EV-METRIC 78% (97th 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 vesicles 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 DG (d)(U)C Protein markers EV: None non-EV: None Proteomics yes EV density (g/ml) 1.14-1.20 Show all info Study aim Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells 3T3-L1 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) 60 Pelleting: rotor type MLA-50 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 60 Wash: Rotor Type MLA-50 Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 31 Lowest density fraction 0.4M Highest density fraction 2M Total gradient volume, incl. sample (mL) 12000 Sample volume (mL) 500 Orientation Top-down Rotor type Not specified Speed (g) 200000 Duration (min) 1080 Fraction volume (mL) 1000 Fraction processing Centrifugation Pelleting: volume per fraction 6000 Pelleting: duration (min) 70 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ Mfge8/ Flotillin2/ TSG101/ Alix/ CD81 Not detected EV-associated proteins caveolin-1/ b-actin Flow cytometry Type of Flow cytometry MACSQuant flow cytometer Calibration bead size 4µm Antibody details provided? No Not detected EV-associated proteins Annexin V Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 100 EV concentration Yes Particle yield EV secreted per adipocyte;Yes, other: 843 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 90-800

	EV170067	1/6	Homo sapiens	HMC-1	DG (d)(U)C	Lässer C	2017	78%
Study summary Full title Two distinct extracellular RNA signatures released by a single cell type identified by microarray and next-generation sequencing. All authors Lässer C, Shelke GV, Yeri A, Kim DK, Crescitelli R, Raimondo S, Sjöstrand M, Gho YS, Van Keuren Jensen K, Lötvall J. Journal RNA Biol Abstract Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in (show more...)Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in many body fluids such as blood, breast milk and cerebrospinal fluid. However, there are conflicting results regarding the nature of exRNA. Here, we have separated 2 distinct exRNA profiles released by mast cells, here termed high-density (HD) and low-density (LD) exRNA. The exRNA in both fractions was characterized by microarray and next-generation sequencing. Both exRNA fractions contained mRNA and miRNA, and the mRNAs in the LD exRNA correlated closely with the cellular mRNA, whereas the HD mRNA did not. Furthermore, the HD exRNA was enriched in lincRNA, antisense RNA, vault RNA, snoRNA, and snRNA with little or no evidence of full-length 18S and 28S rRNA. The LD exRNA was enriched in mitochondrial rRNA, mitochondrial tRNA, tRNA, piRNA, Y RNA, and full-length 18S and 28S rRNA. The proteomes of the HD and LD exRNA-containing fractions were determined with LC-MS/MS and analyzed with Gene Ontology term finder, which showed that both proteomes were associated with the term extracellular vesicles and electron microscopy suggests that at least a part of the exRNA is associated with exosome-like extracellular vesicles. Additionally, the proteins in the HD fractions tended to be associated with the nucleus and ribosomes, whereas the LD fraction proteome tended to be associated with the mitochondrion. We show that the 2 exRNA signatures released by a single cell type can be separated by floatation on a density gradient. These results show that cells can release multiple types of exRNA with substantial differences in RNA species content. This is important for any future studies determining the nature and function of exRNA released from different cells under different conditions. (hide) EV-METRIC 78% (97th 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-like structures 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 DG (d)(U)C Protein markers EV: TSG101/ CD63/ CD81/ Flotillin1/ ANXA2/ CD9 non-EV: Proteomics yes EV density (g/ml) 1.09 - 1.31 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HMC-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g 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 Type 45 Ti Pelleting: speed (g) 120000 Density gradient Type Discontinuous Number of initial discontinuous layers 16 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 35 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 32 Ti Speed (g) 175000 Duration (min) 960 Fraction volume (mL) 3.9 Fraction processing Centrifugation Pelleting: duration (min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 120000 EV-subtype Distinction between multiple subtypes Density Used subtypes 1.09-1.21 g/mL Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ ANXA2/ TSG101/ CD81 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Proteomics database Yes Characterization: RNA analysis RNA analysis Type RNA sequencing;Microarray Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes EM EM-type Immuno-EM EM protein CD63 Image type Close-up, Wide-field Report size (nm) 92

	EV170064	1/2	Bos taurus	Milk	DG (d)(U)C	Samuel, M	2017	78%
Study summary Full title Bovine milk-derived exosomes from colostrum are enriched with proteins implicated in immune response and growth. All authors Samuel M, Chisanga D, Liem M, Keerthikumar S, Anand S, Ang CS, Adda CG, Versteegen E, Jois M, Mathivanan S Journal Abstract Exosomes are extracellular vesicles secreted by multiple cell types into the extracellular space. Th (show more...)Exosomes are extracellular vesicles secreted by multiple cell types into the extracellular space. They contain cell-state specific cargos which often reflects the (patho)physiological condition of the cells/organism. Milk contains high amounts of exosomes and it is unclear whether their cargo is altered based on the lactation stage of the organism. Here, we isolated exosomes from bovine milk that were obtained at various stages of lactation and examined the content by quantitative proteomics. Exosomes were isolated by OptiPrep density gradient centrifugation from milk obtained from cow after 24, 48 and 72 h post calving. As control, exosomes were also isolated from cows during mid-lactation period which has been referred to as mature milk (MM). Biochemical and biophysical characterization of exosomes revealed the high abundance of exosomes in colostrum and MM samples. Quantitative proteomics analysis highlighted the change in the proteomic cargo of exosomes based on the lactation state of the cow. Functional enrichment analysis revealed that exosomes from colostrum are significantly enriched with proteins that can potentially regulate the immune response and growth. This study highlights the importance of exosomes in colostrum and hence opens up new avenues to exploit these vesicles in the regulation of the immune response and growth. (hide) EV-METRIC 78% (85th 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 Milk Sample origin Control condition Focus vesicles 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 DG (d)(U)C Protein markers EV: Alix/ TSG101 non-EV: Albumin/ GM130 Proteomics yes EV density (g/ml) 1.16 Show all info Study aim Identification of content (omics approaches) Sample Species Bos taurus Sample Type Milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 1080 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 0.5 Wash: time (min) 60 Wash: Rotor Type SW 28 Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.5 Orientation Top-down Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins TSG101 Not detected EV-associated proteins Alix Detected contaminants Albumin Not detected contaminants GM130 Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 145 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV170064	2/2	Bos taurus	Milk	DG (d)(U)C	Samuel, M	2017	78%
Study summary Full title Bovine milk-derived exosomes from colostrum are enriched with proteins implicated in immune response and growth. All authors Samuel M, Chisanga D, Liem M, Keerthikumar S, Anand S, Ang CS, Adda CG, Versteegen E, Jois M, Mathivanan S Journal Abstract Exosomes are extracellular vesicles secreted by multiple cell types into the extracellular space. Th (show more...)Exosomes are extracellular vesicles secreted by multiple cell types into the extracellular space. They contain cell-state specific cargos which often reflects the (patho)physiological condition of the cells/organism. Milk contains high amounts of exosomes and it is unclear whether their cargo is altered based on the lactation stage of the organism. Here, we isolated exosomes from bovine milk that were obtained at various stages of lactation and examined the content by quantitative proteomics. Exosomes were isolated by OptiPrep density gradient centrifugation from milk obtained from cow after 24, 48 and 72 h post calving. As control, exosomes were also isolated from cows during mid-lactation period which has been referred to as mature milk (MM). Biochemical and biophysical characterization of exosomes revealed the high abundance of exosomes in colostrum and MM samples. Quantitative proteomics analysis highlighted the change in the proteomic cargo of exosomes based on the lactation state of the cow. Functional enrichment analysis revealed that exosomes from colostrum are significantly enriched with proteins that can potentially regulate the immune response and growth. This study highlights the importance of exosomes in colostrum and hence opens up new avenues to exploit these vesicles in the regulation of the immune response and growth. (hide) EV-METRIC 78% (85th 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 Milk Sample origin Colostrum Focus vesicles 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 DG (d)(U)C Protein markers EV: / Alix/ TSG101 non-EV: Albumin/ GM130 Proteomics yes EV density (g/ml) 1.16 Show all info Study aim Identification of content (omics approaches) Sample Species Bos taurus Sample Type Milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 1080 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 0.5 Wash: time (min) 60 Wash: Rotor Type SW 28 Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.5 Orientation Top-down Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ TSG101 Not detected EV-associated proteins Detected contaminants Albumin Not detected contaminants GM130 Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 125 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV170065	1/1	Homo sapiens	Primary tumor-derived fibroblasts	(d)(U)C	Bhome R	2017	77%
Study summary Full title Exosomal microRNAs derived from colorectal cancer-associated fibroblasts: role in driving cancer progression. All authors Bhome R, Goh RW1, Bullock MD, Pillar N, Thirdborough SM, Mellone M, Mirnezami R, Galea D, Veselkov K, Gu Q, Underwood TJ, Primrose JN, De Wever O, Shomron N, Sayan AE, Mirnezami AH. Journal Aging (Albany NY) Abstract Colorectal cancer is a global disease with increasing incidence. Mortality is largely attributed to (show more...)Colorectal cancer is a global disease with increasing incidence. Mortality is largely attributed to metastatic spread and therefore, a mechanistic dissection of the signals which influence tumor progression is needed. Cancer stroma plays a critical role in tumor proliferation, invasion and chemoresistance. Here, we sought to identify and characterize exosomal microRNAs as mediators of stromal-tumor signaling. In vitro, we demonstrated that fibroblast exosomes are transferred to colorectal cancer cells, with a resultant increase in cellular microRNA levels, impacting proliferation and chemoresistance. To probe this further, exosomal microRNAs were profiled from paired patient-derived normal and cancer-associated fibroblasts, from an ongoing prospective biomarker study. An exosomal cancer-associated fibroblast signature consisting of microRNAs 329, 181a, 199b, 382, 215 and 21 was identified. Of these, miR-21 had highest abundance and was enriched in exosomes. Orthotopic xenografts established with miR-21-overexpressing fibroblasts and CRC cells led to increased liver metastases compared to those established with control fibroblasts. Our data provide a novel stromal exosome signature in colorectal cancer, which has potential for biomarker validation. Furthermore, we confirmed the importance of stromal miR-21 in colorectal cancer progression using an orthotopic model, and propose that exosomes are a vehicle for miR-21 transfer between stromal fibroblasts and cancer cells. (hide) EV-METRIC 77% (97th 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 Colorectal 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 (d)(U)C Adj. k-factor 88.25 (pelleting) / 88.25 (washing) Protein markers EV: Alix/ CD81/ TSG101/ CD63 non-EV: cytochrome c/ GM130/ cytochromec 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 Primary tumor-derived fibroblasts EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 50.3 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 88.25 Wash: time (min) 70 Wash: Rotor Type Type 50.3 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 88.25 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 100 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix, CD63, CD81, TSG101 Not detected contaminants GM130, cytochrome c Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Electron microscopy Extra particle analysis NTA Report type Modus Reported size (nm) 113 EV concentration Yes Particle yield 1570000000000 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV170063	1/2	Homo sapiens	Jurkat	DG (d)(U)C Filtration	Nemeth, Andrea	2017	75%
Study summary Full title Antibiotic-induced release of small extracellular vesicles (exosomes) with surface-associated DNA. All authors Németh A, Orgovan N, Sódar BW, Osteikoetxea X, Pálóczi K, Szabó-Taylor KÉ, Vukman KV, Kittel Á, Turiák L, Wiener Z, Tóth S, Drahos L, Vékey K, Horvath R, Buzás EI. Journal Sci Rep Abstract Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesi (show more...)Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesicles and apoptotic bodies) have attracted substantial attention in various fields of biomedicine. Here we investigated the impact of sustained exposure of cells to the fluoroquinolone antibiotic ciprofloxacin on the released extracellular vesicles. Ciprofloxacin is widely used in humans against bacterial infections as well as in cell cultures against Mycoplasma contamination. However, ciprofloxacin is an inducer of oxidative stress and mitochondrial dysfunction of mammalian cells. Unexpectedly, here we found that ciprofloxacin induced the release of both DNA (mitochondrial and chromosomal sequences) and DNA-binding proteins on the exofacial surfaces of small extracellular vesicles referred to in this paper as exosomes. Furthermore, a label-free optical biosensor analysis revealed DNA-dependent binding of exosomes to fibronectin. DNA release on the surface of exosomes was not affected any further by cellular activation or apoptosis induction. Our results reveal for the first time that prolonged low-dose ciprofloxacin exposure leads to the release of DNA associated with the external surface of exosomes. (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 Cyprofloxacin 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 DG (d)(U)C Filtration Adj. k-factor 115.3 (pelleting) / 115.3 (washing) Protein markers EV: CD63/ HistoneH2B non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Jurkat 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 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 MLA-55 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 115.3 Wash: time (min) 70 Wash: Rotor Type MLA-55 Wash: speed (g) 100000 Wash: adjusted k-factor 115.3 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 0.5 Orientation Top-down (sample migrates downwards) Rotor type MLS-50 Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: duration (min) 180 Pelleting: speed (g) 100000 Filtration steps > 0.45 µm, 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 50-200 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV170063	2/2	Homo sapiens	Jurkat	DG (d)(U)C Filtration	Nemeth, Andrea	2017	75%
Study summary Full title Antibiotic-induced release of small extracellular vesicles (exosomes) with surface-associated DNA. All authors Németh A, Orgovan N, Sódar BW, Osteikoetxea X, Pálóczi K, Szabó-Taylor KÉ, Vukman KV, Kittel Á, Turiák L, Wiener Z, Tóth S, Drahos L, Vékey K, Horvath R, Buzás EI. Journal Sci Rep Abstract Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesi (show more...)Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesicles and apoptotic bodies) have attracted substantial attention in various fields of biomedicine. Here we investigated the impact of sustained exposure of cells to the fluoroquinolone antibiotic ciprofloxacin on the released extracellular vesicles. Ciprofloxacin is widely used in humans against bacterial infections as well as in cell cultures against Mycoplasma contamination. However, ciprofloxacin is an inducer of oxidative stress and mitochondrial dysfunction of mammalian cells. Unexpectedly, here we found that ciprofloxacin induced the release of both DNA (mitochondrial and chromosomal sequences) and DNA-binding proteins on the exofacial surfaces of small extracellular vesicles referred to in this paper as exosomes. Furthermore, a label-free optical biosensor analysis revealed DNA-dependent binding of exosomes to fibronectin. DNA release on the surface of exosomes was not affected any further by cellular activation or apoptosis induction. Our results reveal for the first time that prolonged low-dose ciprofloxacin exposure leads to the release of DNA associated with the external surface of exosomes. (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 DG (d)(U)C Filtration Adj. k-factor 115.3 (pelleting) / 115.3 (washing) Protein markers EV: CD63 non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Jurkat 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 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 MLA-55 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 115.3 Wash: time (min) 70 Wash: Rotor Type MLA-55 Wash: speed (g) 100000 Wash: adjusted k-factor 115.3 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 0.5 Orientation Top-down (sample migrates downwards) Rotor type MLS-50 Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: duration (min) 180 Pelleting: speed (g) 100000 Filtration steps > 0.45 µm, 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 50-200 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV170017	1/1	Mus musculus	Bronchoalveolar lavage fluid	(d)(U)C	Maroto, Rosario	2017	75%
Study summary Full title Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses. All authors Maroto R, Zhao Y, Jamaluddin M, Popov VL, Wang H, Kalubowilage M, Zhang Y, Luisi J, Sun H, Culbertson CT, Bossmann SH, Motamedi M, Brasier AR Journal J Extracell Vesicles Abstract Background: Extracellular vesicles contain biological molecules specified by cell-type of origin and (show more...)Background: Extracellular vesicles contain biological molecules specified by cell-type of origin and modified by microenvironmental changes. To conduct reproducible studies on exosome content and function, storage conditions need to have minimal impact on airway exosome integrity. Aim: We compared surface properties and protein content of airway exosomes that had been freshly isolated vs. those that had been treated with cold storage or freezing. Methods: Mouse bronchoalveolar lavage fluid (BALF) exosomes purified by differential ultracentrifugation were analysed immediately or stored at +4°C or -80°C. Exosomal structure was assessed by dynamic light scattering (DLS), transmission electron microscopy (TEM) and charge density (zeta potential, ζ). Exosomal protein content, including leaking/dissociating proteins, were identified by label-free LC-MS/MS. Results: Freshly isolated BALF exosomes exhibited a mean diameter of 95 nm and characteristic morphology. Storage had significant impact on BALF exosome size and content. Compared to fresh, exosomes stored at +4°C had a 10% increase in diameter, redistribution to polydisperse aggregates and reduced ζ. Storage at -80°C produced an even greater effect, resulting in a 25% increase in diameter, significantly reducing the ζ, resulting in multilamellar structure formation. In fresh exosomes, we identified 1140 high-confidence proteins enriched in 19 genome ontology biological processes. After storage at room temperature, 848 proteins were identified. In preparations stored at +4°C, 224 proteins appeared in the supernatant fraction compared to the wash fractions from freshly prepared exosomes; these proteins represent exosome leakage or dissociation of loosely bound "peri-exosomal" proteins. In preparations stored at -80°C, 194 proteins appeared in the supernatant fraction, suggesting that distinct protein groups leak from exosomes at different storage temperatures. Conclusions: Storage destabilizes the surface characteristics, morphological features and protein content of BALF exosomes. For preservation of the exosome protein content and representative functional analysis, airway exosomes should be analysed immediately after isolation. (hide) EV-METRIC 75% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Bronchoalveolar lavage fluid Sample origin poly IC stimulated Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 60.38 (pelleting) / 60.38 (washing) Protein markers EV: Alix/ CD63,HSP90,Alix/ HSP90/ CD63 non-EV: Grp94,beta-actin/ Grp94/ beta-actin Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Mus musculus Sample Type Bronchoalveolar lavage fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type TLA-100.3 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 60.38 Wash: time (min) 60 Wash: Rotor Type TLA-100.3 Wash: speed (g) 100000 Wash: adjusted k-factor 60.38 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63,HSP90,Alix Not detected contaminants Grp94,beta-actin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Median Reported size (nm) 95 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV170067	2/6	Homo sapiens	HMC-1	DG (d)(U)C Filtration	Lässer C	2017	67%
Study summary Full title Two distinct extracellular RNA signatures released by a single cell type identified by microarray and next-generation sequencing. All authors Lässer C, Shelke GV, Yeri A, Kim DK, Crescitelli R, Raimondo S, Sjöstrand M, Gho YS, Van Keuren Jensen K, Lötvall J. Journal RNA Biol Abstract Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in (show more...)Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in many body fluids such as blood, breast milk and cerebrospinal fluid. However, there are conflicting results regarding the nature of exRNA. Here, we have separated 2 distinct exRNA profiles released by mast cells, here termed high-density (HD) and low-density (LD) exRNA. The exRNA in both fractions was characterized by microarray and next-generation sequencing. Both exRNA fractions contained mRNA and miRNA, and the mRNAs in the LD exRNA correlated closely with the cellular mRNA, whereas the HD mRNA did not. Furthermore, the HD exRNA was enriched in lincRNA, antisense RNA, vault RNA, snoRNA, and snRNA with little or no evidence of full-length 18S and 28S rRNA. The LD exRNA was enriched in mitochondrial rRNA, mitochondrial tRNA, tRNA, piRNA, Y RNA, and full-length 18S and 28S rRNA. The proteomes of the HD and LD exRNA-containing fractions were determined with LC-MS/MS and analyzed with Gene Ontology term finder, which showed that both proteomes were associated with the term extracellular vesicles and electron microscopy suggests that at least a part of the exRNA is associated with exosome-like extracellular vesicles. Additionally, the proteins in the HD fractions tended to be associated with the nucleus and ribosomes, whereas the LD fraction proteome tended to be associated with the mitochondrion. We show that the 2 exRNA signatures released by a single cell type can be separated by floatation on a density gradient. These results show that cells can release multiple types of exRNA with substantial differences in RNA species content. This is important for any future studies determining the nature and function of exRNA released from different cells under different conditions. (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-like structures 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 DG (d)(U)C Filtration Protein markers EV: TSG101/ CD63/ CD81/ Flotillin1/ ANXA2/ CD9 non-EV: Proteomics yes EV density (g/ml) 1.09-1.31 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HMC-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g 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 Type 45 Ti Pelleting: speed (g) 120000 Density gradient Type Discontinuous Number of initial discontinuous layers 16 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 35 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 32 Ti Speed (g) 175000 Duration (min) 960 Fraction volume (mL) 3.9 Fraction processing Centrifugation Pelleting: duration (min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 120000 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes Density Used subtypes 1.24-1.31 g/mL Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ TSG101/ CD81 Not detected EV-associated proteins ANXA2 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Proteomics database Yes Characterization: RNA analysis RNA analysis Type RNA sequencing;Microarray Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes EM EM-type Immuno-EM EM protein CD63 Image type Wide-field Report size (nm) 51

	EV220172	1/2	Homo sapiens	Blood plasma	SEC (non-commercial) UF	Endzeliņš E	2017	63%
Study summary Full title Detection of circulating miRNAs: comparative analysis of extracellular vesicle-incorporated miRNAs and cell-free miRNAs in whole plasma of prostate cancer patients. All authors Endzeliņš E, Berger A, Melne V, Bajo-Santos C, Soboļevska K, Ābols A, Rodriguez M, Šantare D, Rudņickiha A, Lietuvietis V, Llorente A, Linē A Journal BMC Cancer Abstract Circulating cell-free miRNAs have emerged as promising minimally-invasive biomarkers for early detec (show more...)Circulating cell-free miRNAs have emerged as promising minimally-invasive biomarkers for early detection, prognosis and monitoring of cancer. They can exist in the bloodstream incorporated into extracellular vesicles (EVs) and ribonucleoprotein complexes. However, it is still debated if EVs contain biologically meaningful amounts of miRNAs and may provide a better source of miRNA biomarkers than whole plasma. The aim of this study was to systematically compare the diagnostic potential of prostate cancer-associated miRNAs in whole plasma and in plasma EVs. (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 Benign prostatic hyperplasia 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 Size-exclusion chromatography (non-commercial) Ultrafiltration Protein markers EV: CD9/ TSG101/ Actin beta non-EV: Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 3 Kda Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.4 Resin type IsoaddSECResinType 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 CD9/ TSG101/ Actin beta Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment Yes Moment of Proteinase treatment After Proteinase type Proteinase K Proteinase concentration 1000 RNAse treatment Yes RNAse type RNase A RNAse concentration 10 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-150 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Report size (nm) 25-60

	EV220172	2/2	Homo sapiens	Blood plasma	SEC (non-commercial) UF	Endzeliņš E	2017	63%
Study summary Full title Detection of circulating miRNAs: comparative analysis of extracellular vesicle-incorporated miRNAs and cell-free miRNAs in whole plasma of prostate cancer patients. All authors Endzeliņš E, Berger A, Melne V, Bajo-Santos C, Soboļevska K, Ābols A, Rodriguez M, Šantare D, Rudņickiha A, Lietuvietis V, Llorente A, Linē A Journal BMC Cancer Abstract Circulating cell-free miRNAs have emerged as promising minimally-invasive biomarkers for early detec (show more...)Circulating cell-free miRNAs have emerged as promising minimally-invasive biomarkers for early detection, prognosis and monitoring of cancer. They can exist in the bloodstream incorporated into extracellular vesicles (EVs) and ribonucleoprotein complexes. However, it is still debated if EVs contain biologically meaningful amounts of miRNAs and may provide a better source of miRNA biomarkers than whole plasma. The aim of this study was to systematically compare the diagnostic potential of prostate cancer-associated miRNAs in whole plasma and in plasma EVs. (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 Prostate cancer 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 Size-exclusion chromatography (non-commercial) Ultrafiltration Protein markers EV: CD9/ TSG101/ Actin beta non-EV: Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 3 Kda Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.4 Resin type IsoaddSECResinType 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 CD9/ TSG101/ Actin beta Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment Yes Moment of Proteinase treatment After Proteinase type Proteinase K Proteinase concentration 1000 RNAse treatment Yes RNAse type RNase A RNAse concentration 10 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-150 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Report size (nm) 25-60

	EV170061	1/3	Homo sapiens	BEAS2B	(d)(U)C Filtration SEC UF	Benedikter BJ	2017	62%
Study summary Full title Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies. All authors Benedikter BJ, Bouwman FG, Vajen T, Heinzmann ACA, Grauls G, Mariman EC, Wouters EFM, Savelkoul PH, Lopez-Iglesias C, Koenen RR, Rohde GGU, Stassen FRM Journal J Cell Sci Abstract Appropriate isolation methods are essential for unravelling the relative contribution of extracellul (show more...)Appropriate isolation methods are essential for unravelling the relative contribution of extracellular vesicles (EVs) and the EV-free secretome to homeostasis and disease. We hypothesized that ultrafiltration followed by size exclusion chromatography (UF-SEC) provides well-matched concentrates of EVs and free secreted molecules for proteomic and functional studies. Conditioned media of BEAS-2B bronchial epithelial cells were concentrated on 10 kDa centrifuge filters, followed by separation of EVs and free protein using sepharose CL-4B SEC. Alternatively, EVs were isolated by ultracentrifugation. EV recovery was estimated by bead-coupled flow cytometry and tuneable resistive pulse sensing. The proteomic composition of EV isolates and SEC protein fractions was characterized by nano LC-MS/MS. UF-SEC EVs tended to have a higher yield and EV-to-protein rate of purity than ultracentrifugation EVs. UF-SEC EVs and ultracentrifugation EVs showed similar fold-enrichments for biological pathways that were distinct from those of UF-SEC protein. Treatment of BEAS-2B cells with UF-SEC protein, but not with either type of EV isolate increased the IL-8 concentration in the media whereas EVs, but not protein induced monocyte adhesion to endothelial cells. Thus, UF-SEC is a useful alternative for ultracentrifugation and allows comparing the proteomic composition and functional effects of EVs and free secreted molecules. (hide) EV-METRIC 62% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration SEC UF Protein markers EV: CD81/ CD63/ CD9/ MFGE8 non-EV: None Proteomics yes Show all info Study aim Function, Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells BEAS2B EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Sepharose CL-4B Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, CD81, MFGE8 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) CD9, CD63, CD81 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Median Reported size (nm) 61.9 EV concentration Yes Particle yield 3.00E+01 EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 25-200

	EV170054	5/5	Homo sapiens	Urine	(d)(U)C Filtration SEC	Gheinani AH	2017	62%
Study summary Full title Improved isolation strategies to increase the yield and purity of human urinary exosomes for biomarker discovery. All authors Gheinani AH, Vögeli M, Baumgartner U, Vassella E, Draeger A, Burkhard FC, Monastyrskaya K Journal Sci Rep Abstract Circulating miRNAs are detected in extracellular space and body fluids such as urine. Circulating RN (show more...)Circulating miRNAs are detected in extracellular space and body fluids such as urine. Circulating RNAs can be packaged in secreted urinary extracellular vesicles (uEVs) and thus protected from degradation. Urinary exosome preparations might contain specific miRNAs, relevant as biomarkers in renal and bladder diseases. Major difficulties in application of uEVs into the clinical environment are the high variability and low reproducibility of uEV isolation methods. Here we used five different methods to isolate uEVs and compared the size distribution, morphology, yield, presence of exosomal protein markers and RNA content of uEVs. We present an optimized ultracentrifugation and size exclusion chromatography approach for highly reproducible isolation for 50-150 nm uEVs, corresponding to the exosomes, from 50 ml urine. We profiled the miRNA content of uEVs and total urine from the same samples with the NanoString platform and validated the data using qPCR. Our results indicate that 18 miRNAs, robustly detected in uEVs were always present in the total urine. However, 15 miRNAs could be detected only in the total urine preparations and might represent naked circulating miRNA species. This is a novel unbiased and reproducible strategy for uEVs isolation, content normalization and miRNA cargo analysis, suitable for biomarker discovery studies. (hide) EV-METRIC 62% (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 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 (d)(U)C Filtration SEC Protein markers EV: TSG101/ CD81/ TSG101,CD81,CD9/ CD9 non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 23 Sample volume/column (mL) 0.4 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Protein Yield (µg) 6.8 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins TSG101,CD81,CD9 Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mode;mean;size range/distribution;D10;D50;D90 Reported size (nm) 106 EV concentration Yes Particle yield see Fig1A EM EM-type Transmission-EM Image type Close-up, Wide-field Extra information In the optimized protocol, the 16000g pellet was treated with DTT, pelleted and supernatant was pooled with supernatant from the 16000g step to increase yield (see figure 4)

	EV170067	3/6	Homo sapiens	HMC-1	DG (d)(U)C	Lässer C	2017	57%
Study summary Full title Two distinct extracellular RNA signatures released by a single cell type identified by microarray and next-generation sequencing. All authors Lässer C, Shelke GV, Yeri A, Kim DK, Crescitelli R, Raimondo S, Sjöstrand M, Gho YS, Van Keuren Jensen K, Lötvall J. Journal RNA Biol Abstract Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in (show more...)Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in many body fluids such as blood, breast milk and cerebrospinal fluid. However, there are conflicting results regarding the nature of exRNA. Here, we have separated 2 distinct exRNA profiles released by mast cells, here termed high-density (HD) and low-density (LD) exRNA. The exRNA in both fractions was characterized by microarray and next-generation sequencing. Both exRNA fractions contained mRNA and miRNA, and the mRNAs in the LD exRNA correlated closely with the cellular mRNA, whereas the HD mRNA did not. Furthermore, the HD exRNA was enriched in lincRNA, antisense RNA, vault RNA, snoRNA, and snRNA with little or no evidence of full-length 18S and 28S rRNA. The LD exRNA was enriched in mitochondrial rRNA, mitochondrial tRNA, tRNA, piRNA, Y RNA, and full-length 18S and 28S rRNA. The proteomes of the HD and LD exRNA-containing fractions were determined with LC-MS/MS and analyzed with Gene Ontology term finder, which showed that both proteomes were associated with the term extracellular vesicles and electron microscopy suggests that at least a part of the exRNA is associated with exosome-like extracellular vesicles. Additionally, the proteins in the HD fractions tended to be associated with the nucleus and ribosomes, whereas the LD fraction proteome tended to be associated with the mitochondrion. We show that the 2 exRNA signatures released by a single cell type can be separated by floatation on a density gradient. These results show that cells can release multiple types of exRNA with substantial differences in RNA species content. This is important for any future studies determining the nature and function of exRNA released from different cells under different conditions. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Extracellular vesicle-like structures 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 DG (d)(U)C Protein markers EV: non-EV: Proteomics no EV density (g/ml) 1.09 - 1.31 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HMC-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g 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 Type 45 Ti Pelleting: speed (g) 120000 Density gradient Type Discontinuous Number of initial discontinuous layers 16 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 35 Sample volume (mL) 2 Orientation Top-down Rotor type SW 32 Ti Speed (g) 175000 Duration (min) 960 Fraction volume (mL) 3.9 Fraction processing Centrifugation Pelleting: duration (min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 120000 EV-subtype Distinction between multiple subtypes Density Used subtypes 1.09 - 1.21 g/mL Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: RNA analysis RNA analysis Type RNA sequencing,Microarray Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No EM EM-type Immuno-EM EM protein CD63 Image type Close-up, Wide-field Report size (nm) 92

	EV170018	1/12	Homo sapiens	osteoclasts	(d)(U)C Filtration miRCURY Exosome Isolation Kit (Exiqon)	Gebraad A	2017	57%
Study summary Full title Monocyte-derived extracellular vesicles stimulate cytokine secretion and gene expression of matrix metalloproteinases by mesenchymal stem/stromal cells. All authors Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R Journal FEBS J Abstract Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Donor 1, grown on hydroxyapatite 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 Filtration miRCURY Exosome Isolation Kit (Exiqon) Adj. k-factor 211.6 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells osteoclasts EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 211.6 Commercial kit miRCURY Exosome Isolation Kit (Exiqon) Other Name other separation method miRCURY Exosome Isolation Kit (Exiqon) Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 126 EV concentration Yes Particle yield 2.35E+09 particles/ml start sample EM EM-type Transmission-EM Image type Close-up, Wide-field Extra information Pelleting by ultracentrifugation was only added to the protocol for EM analysis.

	EV170018	3/12	Homo sapiens	osteoclasts	(d)(U)C Filtration miRCURY Exosome Isolation Kit (Exiqon)	Gebraad A	2017	57%
Study summary Full title Monocyte-derived extracellular vesicles stimulate cytokine secretion and gene expression of matrix metalloproteinases by mesenchymal stem/stromal cells. All authors Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R Journal FEBS J Abstract Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Donor 2, grown on hydroxyapatite 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 Filtration miRCURY Exosome Isolation Kit (Exiqon) Adj. k-factor 211.6 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells osteoclasts EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 211.6 Commercial kit miRCURY Exosome Isolation Kit (Exiqon) Other Name other separation method miRCURY Exosome Isolation Kit (Exiqon) Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 121 EV concentration Yes Particle yield 6.03E+09 particles/ml start sample EM EM-type Transmission-EM Image type Close-up, Wide-field Extra information Pelleting by ultracentrifugation was only added to the protocol for EM analysis.

	EV170018	5/12	Homo sapiens	osteoclasts	(d)(U)C Filtration miRCURY Exosome Isolation Kit (Exiqon)	Gebraad A	2017	57%
Study summary Full title Monocyte-derived extracellular vesicles stimulate cytokine secretion and gene expression of matrix metalloproteinases by mesenchymal stem/stromal cells. All authors Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R Journal FEBS J Abstract Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Donor 2, grown on polystyrene 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 Filtration miRCURY Exosome Isolation Kit (Exiqon) Adj. k-factor 211.6 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells osteoclasts EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 211.6 Commercial kit miRCURY Exosome Isolation Kit (Exiqon) Other Name other separation method miRCURY Exosome Isolation Kit (Exiqon) Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 126 EV concentration Yes Particle yield 1.96E+10 particles/ml start sample EM EM-type Transmission-EM Image type Close-up, Wide-field Extra information Pelleting by ultracentrifugation was only added to the protocol for EM analysis.

	EV170018	7/12	Homo sapiens	primary circulating monocytes	(d)(U)C Filtration miRCURY Exosome Isolation Kit (Exiqon)	Gebraad A	2017	57%
Study summary Full title Monocyte-derived extracellular vesicles stimulate cytokine secretion and gene expression of matrix metalloproteinases by mesenchymal stem/stromal cells. All authors Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R Journal FEBS J Abstract Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Donor 2, LPS-activated 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 Filtration miRCURY Exosome Isolation Kit (Exiqon) Adj. k-factor 211.6 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary circulating monocytes EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 211.6 Commercial kit miRCURY Exosome Isolation Kit (Exiqon) Other Name other separation method miRCURY Exosome Isolation Kit (Exiqon) Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 91 EV concentration Yes Particle yield 1.68E+09 particles/ml start sample EM EM-type Transmission-EM Image type Close-up, Wide-field Extra information Pelleting by ultracentrifugation was only added to the protocol for EM analysis.

	EV170018	8/12	Homo sapiens	osteoclasts	(d)(U)C Filtration miRCURY Exosome Isolation Kit (Exiqon)	Gebraad A	2017	57%
Study summary Full title Monocyte-derived extracellular vesicles stimulate cytokine secretion and gene expression of matrix metalloproteinases by mesenchymal stem/stromal cells. All authors Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R Journal FEBS J Abstract Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Donor 1, grown on polystyrene 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 Filtration miRCURY Exosome Isolation Kit (Exiqon) Adj. k-factor 211.6 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells osteoclasts EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 211.6 Commercial kit miRCURY Exosome Isolation Kit (Exiqon) Other Name other separation method miRCURY Exosome Isolation Kit (Exiqon) Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 114 EV concentration Yes Particle yield 1.17E+10 particles/ml start sample EM EM-type Transmission-EM Image type Close-up, Wide-field Extra information Pelleting by ultracentrifugation was only added to the protocol for EM analysis.

	EV170018	12/12	Homo sapiens	primary circulating monocytes	(d)(U)C Filtration miRCURY Exosome Isolation Kit (Exiqon)	Gebraad A	2017	57%
Study summary Full title Monocyte-derived extracellular vesicles stimulate cytokine secretion and gene expression of matrix metalloproteinases by mesenchymal stem/stromal cells. All authors Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R Journal FEBS J Abstract Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Donor 1, LPS-activated 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 Filtration miRCURY Exosome Isolation Kit (Exiqon) Adj. k-factor 211.6 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary circulating monocytes EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 211.6 Commercial kit miRCURY Exosome Isolation Kit (Exiqon) Other Name other separation method miRCURY Exosome Isolation Kit (Exiqon) Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 107 EV concentration Yes Particle yield 1.43E+09 particles/ml start sample EM EM-type Transmission-EM Image type Close-up, Wide-field Extra information Pelleting by ultracentrifugation was only added to the protocol for EM analysis.

	EV210397	1/1	Homo sapiens	Mesenchymal stem cells	(d)(U)C Filtration	Pakravan K	2017	56%
Study summary Full title MicroRNA-100 shuttled by mesenchymal stem cell-derived exosomes suppresses in vitro angiogenesis through modulating the mTOR/HIF-1α/VEGF signaling axis in breast cancer cells. All authors Pakravan K, Babashah S, Sadeghizadeh M, Mowla SJ, Mossahebi-Mohammadi M, Ataei F, Dana N, Javan M Journal Cell Oncol (Dordr) Abstract Human mesenchymal stem cells (MSCs) have been shown to be involved in the formation and modulation o (show more...)Human mesenchymal stem cells (MSCs) have been shown to be involved in the formation and modulation of tumor stroma and in interacting with tumor cells, partly through their secretome. Exosomes are nano-sized intraluminal multi-vesicular bodies secreted by most types of cells and have been found to mediate intercellular communication through the transfer of genetic information via coding and non-coding RNAs to recipient cells. Since exosomes are considered as protective and enriched sources of shuttle microRNAs (miRNAs), we hypothesized that exosomal transfer of miRNAs from MSCs may affect tumor cell behavior, particularly angiogenesis. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles 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 Filtration Protein markers EV: CD9 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Mesenchymal stem cells 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 100000 Wash: time (min) 70 Wash: Rotor Type Type 60 Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm 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 Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Modus EM EM-type Scanning-EM Image type Wide-field

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