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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220304	6/30	Homo sapiens	Blood plasma	DG UF SEC (non-commercial)	Dhondt B	2023	100%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (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 Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD81/ Flotillin-1 non-EV: ApoA1/ Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin Proteomics yes EV density (g/ml) 1.09-1.10 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Western Blot Detected EV-associated proteins CD81/ Flotillin-1 Detected contaminants ApoA1 Proteomics database ProteomeXchange Detected contaminants Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins Not detected contaminants Albumin/ GM130/ PMP70/ Prohibitin Characterization: RNA analysis RNA analysis Type RNA -sequencing Database BioProject Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-250 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220090	1/6	Homo sapiens	wound fluid	(d)(U)C Filtration IAF UF DG	Guda PR	2023	100%
Study summary Full title Nanoscopic and Functional Characterization of Keratinocyte-Originating Exosomes in the Wound Fluid of Non-Diabetic and Diabetic Chronic Wound Patients. All authors Guda PR, Sharma A, Anthony AJ, ElMasry MS, Couse AD, Ghatak PD, Das A, Timsina L, Trinidad JC, Roy S, Clemmer DE, Sen CK, Ghatak S Journal Nano Today Abstract Exosomes, a class of extracellular vesicles of endocytic origin, play a critical role in paracrine s (show more...)Exosomes, a class of extracellular vesicles of endocytic origin, play a critical role in paracrine signaling for successful cell-cell crosstalk . However, limitations in our current understanding of these circulating nanoparticles hinder efficient isolation, characterization, and downstream functional analysis of cell-specific exosomes. In this work, we sought to develop a method to isolate and characterize keratinocyte-originated exosomes () from human chronic wound fluid. Furthermore, we studied the significance of in diabetic wounds. LC-MS-MS detection of KRT14 in and subsequent validation by Vesiclepedia and Exocarta databases identified surface KRT14 as a reliable marker of . dSTORM nanoimaging identified KRT14 extracellular vesicles () in human chronic wound fluid, 23% of which were of exosomal origin. An immunomagnetic two-step separation method using KRT14 and tetraspanin antibodies successfully isolated from the heterogeneous pool of EV in chronic wound fluid of 15 non-diabetic and 22 diabetic patients. Isolated (Ø75-150nm) were characterized per EV-track guidelines. dSTORM images, analyzed using online CODI followed by independent validation using Nanometrix, revealed Ø as 80-145nm. The abundance of was low in diabetic wound fluids and negatively correlated with patient HbA1c levels. The isolated from diabetic wound fluid showed a low abundance of small bp RNA (<200 bp). Raman spectroscopy underscored differences in surface lipids between non-diabetic and diabetic Uptake of by monocyte-derived macrophages (MDM) was low for diabetics non-diabetics. Unlike from non-diabetics, the addition of diabetic to MDM polarized with LPS and INFγ resulted in sustained expression of iNOS and pro-inflammatory chemokines known to recruit macrophage (mϕ) This work provides maiden insight into the structure, composition, and function of from chronic wound fluid thus providing a foundation for the study of exosomal malfunction under conditions of diabetic complications such as wound chronicity. (hide) EV-METRIC 100% (75th 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 wound fluid 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 Immunoaffinity capture (non-commercial) Ultrafiltration Density gradient Protein markers EV: HSP90/ Alix/ CD81/ Flotillin-1/ TSG101/ ANXA5/ ICAM/ CD63 non-EV: Prohibitin/ GM130 Proteomics yes EV density (g/ml) 1.15-1.2 Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer/New methodological development/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type wound fluid 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 Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type TLA-120.2 Pelleting: speed (g) 245000 Wash: volume per pellet (ml) 0.5 Wash: time (min) 120 Wash: Rotor Type TLA-120.2 Wash: speed (g) 245000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 0.8M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 0.9 Sample volume (mL) 0.15 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 0.15 Fraction processing Ultracentrifugation Pelleting: volume per fraction 0.5 Pelleting: duration (min) 120 Pelleting: rotor type TLA-120.2 Pelleting: speed (g) 245000 Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Immunoaffinity capture Selected surface protein(s) CD9/CD63/CD81/ KRT14 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per E08 particles: 0.55 Flow cytometry aspecific beads Detected EV-associated proteins HSP90 Not detected contaminants Prohibitin Flow cytometry specific beads Selected surface protein(s) CD9/ CD63/ CD81/ KRT14 Proteomics database No Detected EV-associated proteins Alix/ CD81/ Flotillin-1/ TSG101/ ANXA5/ ICAM Not detected EV-associated proteins CD63/ EPCAM Detected contaminants GM130 Detected EV-associated proteins CD9/C63/CD81/ KRT5 Characterization: RNA analysis RNA analysis Type Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Modus Reported size (nm) 87 EV concentration Yes Particle yield as particles per mg of albumin: 2.30E+08 EM EM-type Scanning-EM/ Immuno-EM EM protein TSG101 Image type Close-up, Wide-field

	EV220024	1/7	Homo sapiens	MDA-MB-231	DG Filtration UF SEC (non-commercial)	Roux, Quentin	2023	100%
Study summary Full title Depletion of soluble cytokines unlocks the immunomodulatory bioactivity of extracellular vesicles All authors Quentin Roux, Robin Boiy, Felix De Vuyst, Mercedes Tkach, Claudio Pinheiro, Sofie de Geyter, Ilkka Miinalainen, Clotilde Théry, Olivier De Wever, An Hendrix Journal J Extracell Vesicles Abstract Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in phys (show more...)Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration. (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 Density gradient Filtration Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: Alix/ CD9/ TSG101/ sCD40L/ EGF/ Eotaxin/ FGF-2/ Flt-3L/ Fractalkine/ G-CSF/ GM-CSF/ GRO_/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-1RA/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-8/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-1/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PDGF-AA/ PDGF-BB/ RANTES/ TGF_/ TNF_ non-EV: Argonaute 2 Proteomics no EV density (g/ml) 1.09-1.11 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDA-MB-231 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 95 Cell count 180000000 Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.8 Sample volume (mL) 0.8 Orientation Bottom-up Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Filtration steps 0.45 µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Alix/ CD9/ TSG101 Not detected contaminants Argonaute 2 Other 1 Luminex Detected EV-associated proteins VEGF-A/ FGF-2/ Fractalkine/ IL-1RA/ GRO_ Not detected EV-associated proteins sCD40L/ EGF/ Eotaxin/ Flt-3L/ G-CSF/ GM-CSF/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PD Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 100 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 50-100

	EV220024	2/7	Homo sapiens	MCF-7	DG Filtration UF SEC (non-commercial)	Roux, Quentin	2023	100%
Study summary Full title Depletion of soluble cytokines unlocks the immunomodulatory bioactivity of extracellular vesicles All authors Quentin Roux, Robin Boiy, Felix De Vuyst, Mercedes Tkach, Claudio Pinheiro, Sofie de Geyter, Ilkka Miinalainen, Clotilde Théry, Olivier De Wever, An Hendrix Journal J Extracell Vesicles Abstract Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in phys (show more...)Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration. (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 Density gradient Filtration Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: Alix/ CD9/ TSG101/ sCD40L/ EGF/ Eotaxin/ FGF-2/ Flt-3L/ Fractalkine/ G-CSF/ GM-CSF/ GRO_/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-1RA/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-8/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-1/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PDGF-AA/ PDGF-BB/ RANTES/ TGF_/ TNF_ non-EV: Argonaute 2 Proteomics no EV density (g/ml) 1.09-1.11 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF-7 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 95 Cell count 179999999 Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.8 Sample volume (mL) 0.8 Orientation Bottom-up Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Filtration steps 0.45 µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Alix/ CD9/ TSG101 Not detected contaminants Argonaute 2 Other 1 Luminex Detected EV-associated proteins VEGF-A/ MCP-1/ FGF-2/ Fractalkine/ IL-1RA/ GRO_ Not detected EV-associated proteins sCD40L/ EGF/ Eotaxin/ Flt-3L/ G-CSF/ GM-CSF/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PD Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 100 EV concentration Yes Particle yield per milliliter of starting sample EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 50-100

	EV220024	4/7	Homo sapiens	Immortalized patient-derived breast CAF	DG Filtration UF SEC (non-commercial)	Roux, Quentin	2023	100%
Study summary Full title Depletion of soluble cytokines unlocks the immunomodulatory bioactivity of extracellular vesicles All authors Quentin Roux, Robin Boiy, Felix De Vuyst, Mercedes Tkach, Claudio Pinheiro, Sofie de Geyter, Ilkka Miinalainen, Clotilde Théry, Olivier De Wever, An Hendrix Journal J Extracell Vesicles Abstract Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in phys (show more...)Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration. (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 Density gradient Filtration Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: Alix/ CD9/ TSG101/ sCD40L/ EGF/ Eotaxin/ FGF-2/ Flt-3L/ Fractalkine/ G-CSF/ GM-CSF/ GRO_/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-1RA/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-8/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-1/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PDGF-AA/ PDGF-BB/ RANTES/ TGF_/ TNF_ non-EV: Argonaute 2 Proteomics no EV density (g/ml) 1.09-1.11 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Immortalized patient-derived breast CAF EV-harvesting Medium Serum free medium Cell viability (%) 95 Cell count 120000000 Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.8 Sample volume (mL) 0.8 Orientation Bottom-up Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Filtration steps 0.45 µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Alix/ CD9/ TSG101 Not detected contaminants Argonaute 2 Other 1 Luminex Detected EV-associated proteins VEGF-A/ MCP-1/ FGF-2/ Fractalkine/ IL-1RA/ GRO_ Not detected EV-associated proteins sCD40L/ EGF/ Eotaxin/ Flt-3L/ G-CSF/ GM-CSF/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-8/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-3/ MDC/ MIP-1_/ MIP- Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 100 EV concentration Yes Particle yield per milliliter of starting sample EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 50-100

	EV230008	4/42	Mus musculus	EO771	(d)(U)C DG UF	Cocozza F	2023	89%
Study summary Full title Extracellular vesicles and co-isolated endogenous retroviruses from murine cancer cells differentially affect dendritic cells. All authors Cocozza F, Martin-Jaular L, Lippens L, Di Cicco A, Arribas YA, Ansart N, Dingli F, Richard M, Merle L, Jouve San Roman M, Poullet P, Loew D, Lévy D, Hendrix A, Kassiotis G, Joliot A, Tkach M, Théry C Journal EMBO J Abstract Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or (show more...)Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or ENPs) that may play a role in intercellular communication. Tumor-derived EVs have been proposed to induce immune priming of antigen presenting cells or to be immuno-suppressive agents. We suspect that such disparate functions are due to variable compositions in EV subtypes and ENPs. We aimed to characterize the array of secreted EVs and ENPs of murine tumor cell lines. Unexpectedly, we identified virus-like particles (VLPs) from endogenous murine leukemia virus in preparations of EVs produced by many tumor cells. We established a protocol to separate small EVs from VLPs and ENPs. We compared their protein composition and analyzed their functional interaction with target dendritic cells. ENPs were poorly captured and did not affect dendritic cells. Small EVs specifically induced dendritic cell death. A mixed large/dense EV/VLP preparation was most efficient to induce dendritic cell maturation and antigen presentation. Our results call for systematic re-evaluation of the respective proportions and functions of non-viral EVs and VLPs produced by murine tumors and their contribution to tumor progression. (hide) EV-METRIC 89% (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 sEV Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Ultrafiltration Protein markers EV: Alix/ CD9/ CD63/ HSP90/ MHC1/ MFGE8 non-EV: Argonaute-2 Proteomics yes EV density (g/ml) 1.015-1.085 Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells EO771 EV-harvesting Medium Serum free medium Cell viability (%) 85 Cell count 100000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: rotor type MLA-80 Pelleting: speed (g) 200000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 6% Highest density fraction 22% Total gradient volume, incl. sample (mL) 16 Sample volume (mL) 1 Orientation Top-down Speed (g) 187000 Duration (min) 90 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 6 Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 2.29E Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose EV-subtype Distinction between multiple subtypes Density Used subtypes 1.015-1.035 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins Alix/ CD9/ CD63/ HSP90/ MHC1/ MFGE8 Detected contaminants Argonaute-2 Proteomics database PRIDE Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 135 EV concentration Yes EM EM-type Cryo-EM Image type Wide-field

	EV230008	5/42	Mus musculus	EO771	(d)(U)C DG UF	Cocozza F	2023	89%
Study summary Full title Extracellular vesicles and co-isolated endogenous retroviruses from murine cancer cells differentially affect dendritic cells. All authors Cocozza F, Martin-Jaular L, Lippens L, Di Cicco A, Arribas YA, Ansart N, Dingli F, Richard M, Merle L, Jouve San Roman M, Poullet P, Loew D, Lévy D, Hendrix A, Kassiotis G, Joliot A, Tkach M, Théry C Journal EMBO J Abstract Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or (show more...)Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or ENPs) that may play a role in intercellular communication. Tumor-derived EVs have been proposed to induce immune priming of antigen presenting cells or to be immuno-suppressive agents. We suspect that such disparate functions are due to variable compositions in EV subtypes and ENPs. We aimed to characterize the array of secreted EVs and ENPs of murine tumor cell lines. Unexpectedly, we identified virus-like particles (VLPs) from endogenous murine leukemia virus in preparations of EVs produced by many tumor cells. We established a protocol to separate small EVs from VLPs and ENPs. We compared their protein composition and analyzed their functional interaction with target dendritic cells. ENPs were poorly captured and did not affect dendritic cells. Small EVs specifically induced dendritic cell death. A mixed large/dense EV/VLP preparation was most efficient to induce dendritic cell maturation and antigen presentation. Our results call for systematic re-evaluation of the respective proportions and functions of non-viral EVs and VLPs produced by murine tumors and their contribution to tumor progression. (hide) EV-METRIC 89% (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 VLP Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Ultrafiltration Protein markers EV: Alix/ CD9/ CD63/ HSP90/ MHC1/ MFGE8 non-EV: Argonaute-2 Proteomics yes EV density (g/ml) 1.015-1.085 Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells EO771 EV-harvesting Medium Serum free medium Cell viability (%) 85 Cell count 100000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: rotor type MLA-80 Pelleting: speed (g) 200000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 6% Highest density fraction 22% Total gradient volume, incl. sample (mL) 16 Sample volume (mL) 1 Orientation Top-down Speed (g) 187000 Duration (min) 90 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 6 Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 2.29E Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose EV-subtype Distinction between multiple subtypes Density Used subtypes 1.065-1.085 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins Alix/ CD9/ CD63/ HSP90/ MHC1/ MFGE8 Not detected contaminants Argonaute-2 Proteomics database PRIDE Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 135 EV concentration Yes EM EM-type Cryo-EM Image type Wide-field

	EV210141	2/5	Homo sapiens	human umbilical vein endothelial cells	IAF Ultrafiltratrion (d)(U)C DG	Zhao F	2023	89%
Study summary Full title Extracellular vesicles from Zika virus-infected cells display viral E protein that binds ZIKV-neutralizing antibodies to prevent infection enhancement. All authors Zhao F, Xu Y, Liu N, Lv D, Chen Y, Liu Z, Jin X, Xiao M, Lavillette D, Zhong J, Bartenschlager R, Long G Journal EMBO J Abstract Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some pa (show more...)Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some parts of the world. Although ZIKV infection is predominantly asymptomatic or associated with mild symptoms, it can lead to neurological complications. ZIKV infection can also cause antibody-dependent enhancement (ADE) of infection with similar viruses, warranting further studies of virion assembly and the function of envelope (E) protein-specific antibodies. Although extracellular vesicles (EVs) from flavivirus-infected cells have been reported to transmit infection, this interpretation is challenged by difficulties in separating EVs from flavivirions due to their similar biochemical composition and biophysical properties. In the present study, a rigorous EV-virion separation method combining sequential ultracentrifugation and affinity capture was developed to study EVs from ZIKV-infected cells. We find that these EVs do not transmit infection, but EVs display abundant E proteins which have an antigenic landscape similar to that of virions carrying E. ZIKV E-coated EVs attenuate antibody-dependent enhancement mediated by ZIKV E-specific and DENV-cross-reactive antibodies in both cell culture and mouse models. We thus report an alternative route for Flavivirus E protein secretion. These results suggest that modulation of E protein release via virions and EVs may present a new approach to regulating flavivirus-host interactions. (hide) EV-METRIC 89% (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 ZIKV infected cells Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Immunoaffinity capture (non-commercial) Ultrafiltratrion (Differential) (ultra)centrifugation Density gradient Protein markers EV: TSG101/ CD63/ CD81/ Alix/ HSP70/ CD9 non-EV: Capsid/ E/ LC3/ Calnexin Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells human umbilical vein endothelial cells EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell count 2,00E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 0% Highest density fraction 80% Sample volume (mL) 1 Orientation Bottom-up Rotor type P55ST Speed (g) 250000 Duration (min) 1080 Fraction volume (mL) 0,3 Ultra filtration Cut-off size (kDa) 100 kDa Membrane type Regenerated cellulose Immunoaffinity capture Selected surface protein(s) CD9 Other Name other separation method Ultrafiltratrion Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD63/ TSG101/ HSP70/ Alix/ CD81 Detected contaminants Capsid/ E Not detected contaminants LC3/ 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 EM EM-type Transmission-EM/ Cryo-EM Image type Wide-field Report size (nm) 100

	EV210141	3/5	Homo sapiens	human umbilical vein endothelial cells	Ultrafiltratrion (d)(U)C DG	Zhao F	2023	89%
Study summary Full title Extracellular vesicles from Zika virus-infected cells display viral E protein that binds ZIKV-neutralizing antibodies to prevent infection enhancement. All authors Zhao F, Xu Y, Liu N, Lv D, Chen Y, Liu Z, Jin X, Xiao M, Lavillette D, Zhong J, Bartenschlager R, Long G Journal EMBO J Abstract Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some pa (show more...)Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some parts of the world. Although ZIKV infection is predominantly asymptomatic or associated with mild symptoms, it can lead to neurological complications. ZIKV infection can also cause antibody-dependent enhancement (ADE) of infection with similar viruses, warranting further studies of virion assembly and the function of envelope (E) protein-specific antibodies. Although extracellular vesicles (EVs) from flavivirus-infected cells have been reported to transmit infection, this interpretation is challenged by difficulties in separating EVs from flavivirions due to their similar biochemical composition and biophysical properties. In the present study, a rigorous EV-virion separation method combining sequential ultracentrifugation and affinity capture was developed to study EVs from ZIKV-infected cells. We find that these EVs do not transmit infection, but EVs display abundant E proteins which have an antigenic landscape similar to that of virions carrying E. ZIKV E-coated EVs attenuate antibody-dependent enhancement mediated by ZIKV E-specific and DENV-cross-reactive antibodies in both cell culture and mouse models. We thus report an alternative route for Flavivirus E protein secretion. These results suggest that modulation of E protein release via virions and EVs may present a new approach to regulating flavivirus-host interactions. (hide) EV-METRIC 89% (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 ZIKV infected cells 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 Ultrafiltratrion (Differential) (ultra)centrifugation Density gradient Protein markers EV: TSG101/ CD63/ CD81/ Alix/ HSP70/ CD9 non-EV: Capsid/ E/ LC3/ Calnexin Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells human umbilical vein endothelial cells EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell count 2,00E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 0% Highest density fraction 80% Sample volume (mL) 1 Orientation Bottom-up Rotor type P55ST Speed (g) 250000 Duration (min) 1080 Fraction volume (mL) 0,3 Ultra filtration Cut-off size (kDa) 100 kDa Membrane type Regenerated cellulose Other Name other separation method Ultrafiltratrion Characterization: Protein analysis Western Blot Detected EV-associated proteins CD9/ CD63/ TSG101/ HSP70/ Alix/ CD81 Detected contaminants Capsid/ E Not detected contaminants LC3/ 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 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 100

	EV210215	3/4	Homo sapiens	PBS spiked with recombinant EV (gag-EGFP HEK293T)	DG	Van Dorpe S	2023	88%
Study summary Full title Integrating automated liquid handling in the separation workflow of extracellular vesicles enhances specificity and reproducibility. All authors Van Dorpe S, Lippens L, Boiy R, Pinheiro C, Vergauwen G, Rappu P, Miinalainen I, Tummers P, Denys H, De Wever O, Hendrix A Journal J Nanobiotechnology Abstract Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for (show more...)Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for numerous diseases. Major impediments of EV-based biomarker discovery include the specificity and reproducibility of EV sample preparation as well as intensive manual labor. We present an automated liquid handling workstation for the density-based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers. (hide) EV-METRIC 88% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type PBS spiked with recombinant EV (gag-EGFP HEK293T) Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient Protein markers EV: TSG101/ CD81/ Alix/ p24/ CD9/ syntenin-1 non-EV: None Proteomics no EV density (g/ml) 1.086-1.119 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type PBS spiked with recombinant EV (gag-EGFP HEK293T) Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Size-exclusion chromatography Resin type Characterization: Protein analysis Protein Concentration Method Not determined Protein Yield (µg) as percentage of spiked rEV ELISA Detected EV-associated proteins p24 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 140 EV concentration Yes Particle yield as percentage of spiked rEV EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) Not reported

	EV210215	4/4	Homo sapiens	PBS spiked with recombinant EV (gag-EGFP HEK293T)	DG	Van Dorpe S	2023	88%
Study summary Full title Integrating automated liquid handling in the separation workflow of extracellular vesicles enhances specificity and reproducibility. All authors Van Dorpe S, Lippens L, Boiy R, Pinheiro C, Vergauwen G, Rappu P, Miinalainen I, Tummers P, Denys H, De Wever O, Hendrix A Journal J Nanobiotechnology Abstract Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for (show more...)Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for numerous diseases. Major impediments of EV-based biomarker discovery include the specificity and reproducibility of EV sample preparation as well as intensive manual labor. We present an automated liquid handling workstation for the density-based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers. (hide) EV-METRIC 88% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type PBS spiked with recombinant EV (gag-EGFP HEK293T) Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient Protein markers EV: TSG101/ CD81/ Alix/ p24/ CD9/ syntenin-1 non-EV: None Proteomics no EV density (g/ml) 1.086-1.119 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type PBS spiked with recombinant EV (gag-EGFP HEK293T) Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 0.8 Fraction processing None Characterization: Protein analysis Protein Concentration Method Not determined ELISA Detected EV-associated proteins p24 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 140 EV concentration Yes Particle yield as percentage of spiked rEV EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) Not reported

	EV210215	1/4	Homo sapiens	Blood plasma	(d)(U)C DG UF SEC (non-commercial)	Van Dorpe S	2023	86%
Study summary Full title Integrating automated liquid handling in the separation workflow of extracellular vesicles enhances specificity and reproducibility. All authors Van Dorpe S, Lippens L, Boiy R, Pinheiro C, Vergauwen G, Rappu P, Miinalainen I, Tummers P, Denys H, De Wever O, Hendrix A Journal J Nanobiotechnology Abstract Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for (show more...)Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for numerous diseases. Major impediments of EV-based biomarker discovery include the specificity and reproducibility of EV sample preparation as well as intensive manual labor. We present an automated liquid handling workstation for the density-based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers. (hide) EV-METRIC 86% (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 Blood plasma 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 (Differential) (ultra)centrifugation Density gradient Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: Albumin/ ApoA1/ ApoB Proteomics yes EV density (g/ml) 1.086-1.119 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma 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 No Density gradient 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 Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 15 Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 12 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Proteomics database ProteomeXchange Consortium Detected contaminants Albumin/ ApoA1/ ApoB Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-150

	EV210215	2/4	Homo sapiens	urine	(d)(U)C DG UF	Van Dorpe S	2023	86%
Study summary Full title Integrating automated liquid handling in the separation workflow of extracellular vesicles enhances specificity and reproducibility. All authors Van Dorpe S, Lippens L, Boiy R, Pinheiro C, Vergauwen G, Rappu P, Miinalainen I, Tummers P, Denys H, De Wever O, Hendrix A Journal J Nanobiotechnology Abstract Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for (show more...)Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for numerous diseases. Major impediments of EV-based biomarker discovery include the specificity and reproducibility of EV sample preparation as well as intensive manual labor. We present an automated liquid handling workstation for the density-based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers. (hide) EV-METRIC 86% (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 urine Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Ultrafiltration Protein markers EV: None non-EV: Albumin/ Tamm-Horsfall protein Proteomics yes EV density (g/ml) 1.086-1.119 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 Between 800 g and 10,000 g Pelleting performed No Density gradient 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 Bottom-up Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 15 Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 12 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Proteomics database ProteomeXchange Consortium Detected contaminants Albumin/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-150

	EV220304	3/30	Homo sapiens	Blood plasma	DG UF SEC (non-commercial)	Dhondt B	2023	83%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 83% (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 Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin Proteomics yes EV density (g/ml) 1.09-1.10 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Proteomics database ProteomeXchange Detected contaminants Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins Not detected contaminants Albumin/ Calreticulin/ GM130/ PMP70/ Prohibitin Characterization: RNA analysis RNA analysis Type RNA -sequencing Database BioProject Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-250 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV220304	9/30	Homo sapiens	Blood plasma	DG UF SEC (non-commercial)	Dhondt B	2023	83%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 83% (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 Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin Proteomics yes EV density (g/ml) 1.09-1.10 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Proteomics database ProteomeXchange Detected contaminants Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins Not detected contaminants Albumin/ GM130/ PMP70/ Prohibitin Characterization: RNA analysis RNA analysis Type RNA -sequencing Database BioProject Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-250 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV220304	12/30	Homo sapiens	Serum	DG UF SEC (non-commercial)	Dhondt B	2023	83%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 83% (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 Serum Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin Proteomics yes EV density (g/ml) 1.09-1.10 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species Homo sapiens Sample Type Serum Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Proteomics database ProteomeXchange Detected contaminants Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins Not detected contaminants Albumin/ GM130/ PMP70/ Prohibitin Characterization: RNA analysis RNA analysis Type RNA -sequencing Database BioProject Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-250 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV220304	15/30	NA	NA	DG UF SEC (non-commercial)	Dhondt B	2023	83%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 83% (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 NA Sample origin NA Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin Proteomics yes EV density (g/ml) 1.09-1.10 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Proteomics database ProteomeXchange Detected contaminants Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins Not detected contaminants Albumin/ GM130/ PMP70/ Prohibitin Characterization: RNA analysis RNA analysis Type RNA -sequencing Database BioProject Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-250 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV220304	18/30	NA	NA	DG UF SEC (non-commercial)	Dhondt B	2023	83%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 83% (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 NA Sample origin NA Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin Proteomics yes EV density (g/ml) 1.09-1.10 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Proteomics database ProteomeXchange Detected contaminants Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins Not detected contaminants Albumin/ GM130/ PMP70/ Prohibitin Characterization: RNA analysis RNA analysis Type RNA -sequencing Database BioProject Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-250 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV220304	21/30	NA	NA	DG UF SEC (non-commercial)	Dhondt B	2023	83%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 83% (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 NA Sample origin NA Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin Proteomics yes EV density (g/ml) 1.09-1.10 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Proteomics database ProteomeXchange Detected contaminants Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins Not detected contaminants Albumin/ GM130/ PMP70/ Prohibitin Characterization: RNA analysis RNA analysis Type RNA -sequencing Database BioProject Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-250 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV220304	24/30	NA	NA	DG UF SEC (non-commercial)	Dhondt B	2023	83%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 83% (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 NA Sample origin NA Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin Proteomics yes EV density (g/ml) 1.09-1.10 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Proteomics database ProteomeXchange Detected contaminants Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins Not detected contaminants Albumin/ GM130/ PMP70/ Prohibitin Characterization: RNA analysis RNA analysis Type RNA -sequencing Database BioProject Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-250 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV220304	30/30	NA	NA	DG UF SEC (non-commercial)	Dhondt B	2023	83%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 83% (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 NA Sample origin NA Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin Proteomics yes EV density (g/ml) 1.09-1.10 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Proteomics database ProteomeXchange Detected contaminants Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins Not detected contaminants Albumin/ GM130/ PMP70/ Prohibitin Characterization: RNA analysis RNA analysis Type RNA -sequencing Database BioProject Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-250 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV231018	1/2	Homo sapiens	Wharton's jelly MSC	(d)(U)C qEVsingle Gen2	Tscherrig V	2023	78%
Study summary Full title MicroRNA Cargo in Wharton's Jelly MSC Small Extracellular Vesicles: Key Functionality to In Vitro Prevention and Treatment of Premature White Matter Injury. All authors Tscherrig V, Cottagnoud S, Haesler V, Renz P, Surbek D, Schoeberlein A, Joerger-Messerli MS Journal Stem Cell Rev Rep Abstract Preterm birth is the leading cause of childhood morbidity and mortality and can result in white matt (show more...)Preterm birth is the leading cause of childhood morbidity and mortality and can result in white matter injury (WMI), leading to long-term neurological disabilities with global health burden. Mesenchymal stromal cell-derived small extracellular vesicles (MSC-sEV) are a promising therapeutic agent for treating perinatal neurological injury. They carry microRNAs (miRNAs) predicted to be involved in the onset of premature WMI. We hypothesize that miRNAs have a key function in the beneficial effects of MSC-sEV. We isolated MSC from umbilical cord tissue, the Wharton's jelly (WJ), and purified small extracellular vesicles (sEV) from WJ-MSC culture supernatant by ultracentrifugation and size exclusion chromatography. The miRNA content was quantified by real-time polymerase chain reaction. A luciferase gene assay validated silencing of TP53 and TAOK1, which we previously identified as predicted target genes of MSC-sEV miRNAs by Next Generation Sequencing and pathway enrichment analysis. The impact of sEV miRNAs on oligodendroglial maturation and neuronal apoptosis was evaluated using an in vitro oxygen-glucose deprivation model (OGD/R) by knocking-down DROSHA in WJ-MSC, which initiates miRNA processing. WJ-MSC-sEV contained miRNAs involved in WMI, namely hsa-miR-22-3p, hsa-miR-21-5p, hsa-miR-27b-3p, and the hsa-let-7 family. The luciferase assay strongly indicated an inhibitory effect of sEV miRNAs on the gene expression of TP53 and TAOK1. Small EV initiated oligodendrocyte maturation and reduced OGD/R-mediated neuronal apoptosis. Knocking-down DROSHA in WJ-MSC reduced the expression of sEV miRNAs and led to the loss of their beneficial effects. Our in vitro study strongly indicates the key function of miRNAs in the therapeutic potential of WJ-MSC-sEV in premature WMI. (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 (Differential) (ultra)centrifugation Commercial method Protein markers EV: CD63/ CD81/ Syntenin/ CD9 non-EV: GM130/ Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Wharton's jelly MSC 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: rotor type TLA-100.3 Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 1.5 Wash: time (min) 70 Wash: Rotor Type TLA-100.3 Wash: speed (g) 110000 Commercial kit qEVsingle Gen2 Characterization: Protein analysis Protein Concentration Method NanoView Spectrometry Protein Yield (µg) per microliter of recovered sample Western Blot Detected EV-associated proteins CD63/ CD81/ Syntenin Not detected contaminants GM130/ Calnexin Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 148 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.00E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV231018	2/2	Homo sapiens	Wharton's jelly MSC	(d)(U)C qEVsingle Gen2	Tscherrig V	2023	78%
Study summary Full title MicroRNA Cargo in Wharton's Jelly MSC Small Extracellular Vesicles: Key Functionality to In Vitro Prevention and Treatment of Premature White Matter Injury. All authors Tscherrig V, Cottagnoud S, Haesler V, Renz P, Surbek D, Schoeberlein A, Joerger-Messerli MS Journal Stem Cell Rev Rep Abstract Preterm birth is the leading cause of childhood morbidity and mortality and can result in white matt (show more...)Preterm birth is the leading cause of childhood morbidity and mortality and can result in white matter injury (WMI), leading to long-term neurological disabilities with global health burden. Mesenchymal stromal cell-derived small extracellular vesicles (MSC-sEV) are a promising therapeutic agent for treating perinatal neurological injury. They carry microRNAs (miRNAs) predicted to be involved in the onset of premature WMI. We hypothesize that miRNAs have a key function in the beneficial effects of MSC-sEV. We isolated MSC from umbilical cord tissue, the Wharton's jelly (WJ), and purified small extracellular vesicles (sEV) from WJ-MSC culture supernatant by ultracentrifugation and size exclusion chromatography. The miRNA content was quantified by real-time polymerase chain reaction. A luciferase gene assay validated silencing of TP53 and TAOK1, which we previously identified as predicted target genes of MSC-sEV miRNAs by Next Generation Sequencing and pathway enrichment analysis. The impact of sEV miRNAs on oligodendroglial maturation and neuronal apoptosis was evaluated using an in vitro oxygen-glucose deprivation model (OGD/R) by knocking-down DROSHA in WJ-MSC, which initiates miRNA processing. WJ-MSC-sEV contained miRNAs involved in WMI, namely hsa-miR-22-3p, hsa-miR-21-5p, hsa-miR-27b-3p, and the hsa-let-7 family. The luciferase assay strongly indicated an inhibitory effect of sEV miRNAs on the gene expression of TP53 and TAOK1. Small EV initiated oligodendrocyte maturation and reduced OGD/R-mediated neuronal apoptosis. Knocking-down DROSHA in WJ-MSC reduced the expression of sEV miRNAs and led to the loss of their beneficial effects. Our in vitro study strongly indicates the key function of miRNAs in the therapeutic potential of WJ-MSC-sEV in premature WMI. (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 DROSHA siRNA modified 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 (Differential) (ultra)centrifugation Commercial method Protein markers EV: CD63/ CD81/ Syntenin non-EV: GM130/ Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Wharton's jelly MSC 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: rotor type TLA-100.3 Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 1.5 Wash: time (min) 70 Wash: Rotor Type TLA-100.3 Wash: speed (g) 110000 Commercial kit qEVsingle Gen2 Characterization: Protein analysis Protein Concentration Method NanoView Spectrometry Protein Yield (µg) per microliter of recovered sample Western Blot Detected EV-associated proteins CD63/ CD81/ Syntenin Not detected contaminants GM130/ Calnexin Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment Yes Moment of Proteinase treatment After Proteinase type Proteinase K Proteinase concentration 2000 RNAse treatment Yes RNAse type RNase A RNAse concentration 0.02 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 148 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.00E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220429	1/1	Mus musculus	Osteoclasts	(d)(U)C	Faqeer, Abdullah	2023	78%
Study summary Full title Cleaved SPP1-rich extracellular vesicles from osteoclasts promote bone regeneration via TGFβ1/SMAD3 signaling All authors Abdullah Faqeer, Mengzhen Wang, Gulzar Alam, Arshad Ahmed Padhiar, Dexiu Zheng, Zhiming Luo, Irene Shuping Zhao, Guangqian Zhou, Jeroen van den Beucken, Huanan Wang, Yang Zhang Journal Biomaterials Abstract Bone remodeling is a tightly coupled process between bone forming osteoblasts (OBs) and bone resorbi (show more...)Bone remodeling is a tightly coupled process between bone forming osteoblasts (OBs) and bone resorbing osteoclasts (OCs) to maintain bone architecture and systemic mineral homeostasis throughout life. However, the mechanisms responsible for the coupling between OCs and OBs have not been fully elucidated. Herein, we first validate that secreted extracellular vesicles by osteoclasts (OC-EVs) promote osteogenic differentiation of mesenchymal stem cells (MSCs) and further demonstrate the efficacy of osteoclasts and their secreted EVs in treating tibial bone defects. Furthermore, we show that OC-EVs contain several osteogenesis-promoting proteins as cargo. By employing proteomic and functional analysis, we reveal that mature osteoclasts secrete thrombin cleaved phosphoprotein 1 (SPP1) through extracellular vesicles which triggers MSCs osteogenic differentiation into OBs by activating Transforming Growth Factor β1 (TGFβ1) and Smad family member 3 (SMAD3) signaling. In conclusion, our findings prove an important role of SPP1, present as cargo in OC-derived EVs, in signaling to MSCs and driving their differentiation into OBs. This biological mechanism implies a paradigm shift regarding the role of osteoclasts and their signaling toward the treatment of skeletal disorders which require bone formation. (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 Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: HSPA8/ CD9/ CD81/ TSG101/ beta actin non-EV: calnexin Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Osteoclasts EV-harvesting Medium Serum free medium Cell count 3000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 8 Wash: time (min) 60 Wash: Rotor Type Type 70.1Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins HSPA8/ CD9/ CD81/ TSG101/ beta actin Not detected contaminants calnexin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 180 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 155

	EV220369	1/5	Homo sapiens	Blood plasma	(d)(U)C	Lapin M	2023	78%
Study summary Full title Extracellular vesicles as a potential source of tumor-derived DNA in advanced pancreatic cancer. All authors Lapin M, Tjensvoll K, Nedrebø K, Taksdal E, Janssen H, Gilje B, Nordgård O Journal PLoS One Abstract Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Sev (show more...)Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Several studies have highlighted the potential of EV-derived DNA (evDNA) as a circulating biomarker, even demonstrating that evDNA can outperform cell-free DNA (cfDNA) in terms of sensitivity. Here, we evaluated EVs as a potential source of tumor-derived DNA in patients with advanced pancreatic cancer. evDNA from both DNase-treated and untreated EV samples was analyzed to determine whether the DNA was primarily located internally or outside (surface-bound) the EVs. To assess whether methodology affected the results, we isolated EVs using four different methods for small EV isolation and differential centrifugation for isolating large EVs. Our results indicated that the DNA content of EVs was significantly less than the cfDNA content isolated from the same plasma volume (p < 0.001). Most of the detected evDNA was also located on the outside of the vesicles. Furthermore, the fraction of tumor-derived DNA in EVs was similar to that found in cfDNA. In conclusion, our results suggest that quantification of evDNA, as a source of tumor-derived DNA, does not add information to that obtained with cfDNA, at least not in patients with advanced pancreatic cancer. (hide) EV-METRIC 78% (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 Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD9/ CD63/ CD81/ TSG101 non-EV: ApoA1 Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma 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: rotor type Type 70 Ti Pelleting: speed (g) 20 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD9/ CD63/ CD81/ TSG101 Detected contaminants ApoA1 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 1-10000 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) Equal or larger than 200nm

	EV220369	2/5	Homo sapiens	Blood plasma	(d)(U)C Filtration qEV UF	Lapin M	2023	78%
Study summary Full title Extracellular vesicles as a potential source of tumor-derived DNA in advanced pancreatic cancer. All authors Lapin M, Tjensvoll K, Nedrebø K, Taksdal E, Janssen H, Gilje B, Nordgård O Journal PLoS One Abstract Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Sev (show more...)Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Several studies have highlighted the potential of EV-derived DNA (evDNA) as a circulating biomarker, even demonstrating that evDNA can outperform cell-free DNA (cfDNA) in terms of sensitivity. Here, we evaluated EVs as a potential source of tumor-derived DNA in patients with advanced pancreatic cancer. evDNA from both DNase-treated and untreated EV samples was analyzed to determine whether the DNA was primarily located internally or outside (surface-bound) the EVs. To assess whether methodology affected the results, we isolated EVs using four different methods for small EV isolation and differential centrifugation for isolating large EVs. Our results indicated that the DNA content of EVs was significantly less than the cfDNA content isolated from the same plasma volume (p < 0.001). Most of the detected evDNA was also located on the outside of the vesicles. Furthermore, the fraction of tumor-derived DNA in EVs was similar to that found in cfDNA. In conclusion, our results suggest that quantification of evDNA, as a source of tumor-derived DNA, does not add information to that obtained with cfDNA, at least not in patients with advanced pancreatic cancer. (hide) EV-METRIC 78% (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 Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Commercial method Ultrafiltration Protein markers EV: CD9/ CD63/ CD81/ TSG101 non-EV: ApoA1 Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma 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 No Filtration steps Larger than 0.45 µm Ultra filtration Cut-off size (kDa) 100 kDa Membrane type Regenerated cellulose Commercial kit qEV Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD9/ CD63/ CD81/ TSG101 Detected contaminants ApoA1 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 1-10000 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) Below or equal to 200nm

	EV220369	3/5	Homo sapiens	Blood plasma	(d)(U)C Filtration ExoEasy	Lapin M	2023	78%
Study summary Full title Extracellular vesicles as a potential source of tumor-derived DNA in advanced pancreatic cancer. All authors Lapin M, Tjensvoll K, Nedrebø K, Taksdal E, Janssen H, Gilje B, Nordgård O Journal PLoS One Abstract Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Sev (show more...)Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Several studies have highlighted the potential of EV-derived DNA (evDNA) as a circulating biomarker, even demonstrating that evDNA can outperform cell-free DNA (cfDNA) in terms of sensitivity. Here, we evaluated EVs as a potential source of tumor-derived DNA in patients with advanced pancreatic cancer. evDNA from both DNase-treated and untreated EV samples was analyzed to determine whether the DNA was primarily located internally or outside (surface-bound) the EVs. To assess whether methodology affected the results, we isolated EVs using four different methods for small EV isolation and differential centrifugation for isolating large EVs. Our results indicated that the DNA content of EVs was significantly less than the cfDNA content isolated from the same plasma volume (p < 0.001). Most of the detected evDNA was also located on the outside of the vesicles. Furthermore, the fraction of tumor-derived DNA in EVs was similar to that found in cfDNA. In conclusion, our results suggest that quantification of evDNA, as a source of tumor-derived DNA, does not add information to that obtained with cfDNA, at least not in patients with advanced pancreatic cancer. (hide) EV-METRIC 78% (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 Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Commercial method Protein markers EV: CD9/ CD63/ CD81/ TSG101 non-EV: ApoA1 Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma 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 No Filtration steps Larger than 0.45 µm Commercial kit ExoEasy Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD9/ CD63/ CD81/ TSG101 Detected contaminants ApoA1 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 1-10000 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) Below or equal to 200nm

	EV220369	5/5	Homo sapiens	Blood plasma	(d)(U)C Filtration	Lapin M	2023	78%
Study summary Full title Extracellular vesicles as a potential source of tumor-derived DNA in advanced pancreatic cancer. All authors Lapin M, Tjensvoll K, Nedrebø K, Taksdal E, Janssen H, Gilje B, Nordgård O Journal PLoS One Abstract Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Sev (show more...)Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Several studies have highlighted the potential of EV-derived DNA (evDNA) as a circulating biomarker, even demonstrating that evDNA can outperform cell-free DNA (cfDNA) in terms of sensitivity. Here, we evaluated EVs as a potential source of tumor-derived DNA in patients with advanced pancreatic cancer. evDNA from both DNase-treated and untreated EV samples was analyzed to determine whether the DNA was primarily located internally or outside (surface-bound) the EVs. To assess whether methodology affected the results, we isolated EVs using four different methods for small EV isolation and differential centrifugation for isolating large EVs. Our results indicated that the DNA content of EVs was significantly less than the cfDNA content isolated from the same plasma volume (p < 0.001). Most of the detected evDNA was also located on the outside of the vesicles. Furthermore, the fraction of tumor-derived DNA in EVs was similar to that found in cfDNA. In conclusion, our results suggest that quantification of evDNA, as a source of tumor-derived DNA, does not add information to that obtained with cfDNA, at least not in patients with advanced pancreatic cancer. (hide) EV-METRIC 78% (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 Blood plasma Sample origin Pancreatic 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 (Differential) (ultra)centrifugation Filtration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 32 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100,000 Filtration steps Larger than 0.45 µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63/ CD81/ TSG101 Detected contaminants ApoA1 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 1-10000 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) Below or equal to 200nm

	EV220024	3/7	Homo sapiens	Immortalized patient-derived breast CAF	(d)(U)C	Roux, Quentin	2023	78%
Study summary Full title Depletion of soluble cytokines unlocks the immunomodulatory bioactivity of extracellular vesicles All authors Quentin Roux, Robin Boiy, Felix De Vuyst, Mercedes Tkach, Claudio Pinheiro, Sofie de Geyter, Ilkka Miinalainen, Clotilde Théry, Olivier De Wever, An Hendrix Journal J Extracell Vesicles Abstract Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in phys (show more...)Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration. (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 Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD9/ TSG101/ sCD40L/ EGF/ Eotaxin/ FGF-2/ Flt-3L/ Fractalkine/ G-CSF/ GM-CSF/ GRO_/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-1RA/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-8/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-1/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PDGF-AA/ PDGF-BB/ RANTES/ TGF_/ TNF_ non-EV: Argonaute 2 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Immortalized patient-derived breast CAF EV-harvesting Medium Serum free medium Cell viability (%) 95 Cell count 120000000 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 SW 32.1 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 16 Wash: time (min) 60 Wash: Rotor Type SW 32.1 Ti Wash: speed (g) 100000 Size-exclusion chromatography Resin type Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD9/ TSG101 Detected contaminants Argonaute 2 Other 1 Luminex Detected EV-associated proteins VEGF-A/ MCP-1/ FGF-2/ Fractalkine/ IL-1RA/ PDGF-AA/ IL-8/ GRO_ Not detected EV-associated proteins sCD40L/ EGF/ Eotaxin/ Flt-3L/ G-CSF/ GM-CSF/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PD Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 100 EV concentration Yes Particle yield per milliliter of starting sample EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 50-100

	EV220024	5/7	Homo sapiens	Immortalized patient-derived breast CAF	Filtration UF SEC (non-commercial)	Roux, Quentin	2023	78%
Study summary Full title Depletion of soluble cytokines unlocks the immunomodulatory bioactivity of extracellular vesicles All authors Quentin Roux, Robin Boiy, Felix De Vuyst, Mercedes Tkach, Claudio Pinheiro, Sofie de Geyter, Ilkka Miinalainen, Clotilde Théry, Olivier De Wever, An Hendrix Journal J Extracell Vesicles Abstract Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in phys (show more...)Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration. (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 Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Filtration Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: Alix/ CD9/ TSG101/ sCD40L/ EGF/ Eotaxin/ FGF-2/ Flt-3L/ Fractalkine/ G-CSF/ GM-CSF/ GRO_/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-1RA/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-8/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-1/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PDGF-AA/ PDGF-BB/ RANTES/ TGF_/ TNF_ non-EV: Argonaute 2 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Immortalized patient-derived breast CAF EV-harvesting Medium Serum free medium Cell viability (%) 95 Cell count 120000000 Separation Method Filtration steps 0.45 µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD9/ TSG101 Not detected contaminants Argonaute 2 Other 1 Luminex Detected EV-associated proteins to complete Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 100 EV concentration Yes Particle yield per milliliter of starting sample EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 50-100

	EV220024	6/7	Homo sapiens	MDA-MB-231	(d)(U)C	Roux, Quentin	2023	78%
Study summary Full title Depletion of soluble cytokines unlocks the immunomodulatory bioactivity of extracellular vesicles All authors Quentin Roux, Robin Boiy, Felix De Vuyst, Mercedes Tkach, Claudio Pinheiro, Sofie de Geyter, Ilkka Miinalainen, Clotilde Théry, Olivier De Wever, An Hendrix Journal J Extracell Vesicles Abstract Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in phys (show more...)Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration. (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 Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD9/ TSG101 non-EV: Argonaute 2 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDA-MB-231 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 95 Cell count 180000000 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 SW 32.1 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 16 Wash: time (min) 60 Wash: Rotor Type SW 32.1 Ti Wash: speed (g) 100000 Size-exclusion chromatography Resin type Characterization: Protein analysis Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Alix/ CD9/ TSG101 Detected contaminants Argonaute 2 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 100 EV concentration Yes Particle yield per milliliter of starting sample EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 50-100

	EV210382	1/4	Homo sapiens	maternal placenta perfusate	(d)(U)C	Awoyemi T	2023	78%
Study summary Full title A cross-sectional analysis of syncytiotrophoblast membrane extracellular vesicles-derived transcriptomic biomarkers in early-onset preeclampsia. All authors Awoyemi T, Zhang W, Rahbar M, Cribbs A, Logenthiran P, Jiang S, Collett G, Cerdeira AS, Vatish M Journal Front Cardiovasc Med Abstract Preeclampsia (PE) is a pregnancy-specific hypertensive disorder affecting 2%-8% of pregnancies world (show more...)Preeclampsia (PE) is a pregnancy-specific hypertensive disorder affecting 2%-8% of pregnancies worldwide. Biomarker(s) for the disorder exists, but while these have excellent negative predictive value, their positive predictive value is poor. Extracellular vesicles released by the placenta into the maternal circulation, syncytiotrophoblast membrane extracellular vesicles (STB-EVs), have been identified as being involved in PE with the potential to act as liquid biopsies. (hide) EV-METRIC 78% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type maternal placenta perfusate Sample origin Control condition Focus vesicles medium/large extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Alix/ PLAP/ CD9 non-EV: Cytochrome C Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type maternal placenta perfusate 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) 30 Pelleting: rotor type Sorvall TST28.39 Pelleting: speed (g) 10,000 Wash: volume per pellet (ml) 45 Wash: time (min) 30 Wash: Rotor Type Sorvall TST28.39 Wash: speed (g) 10,000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 8680-10240 Western Blot Detected EV-associated proteins Alix/ PLAP Not detected EV-associated proteins CD9 Not detected contaminants Cytochrome C Proteomics database PRIDE Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR/ RNA-sequencing/ Capillary electrophoresis (e.g. Bioanalyzer) Database NCBI geo Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 205.8 EV concentration Yes Particle yield 6.98E11-1.12E12 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210382	2/4	Homo sapiens	maternal placenta perfusate	(d)(U)C Filtration	Awoyemi T	2023	78%
Study summary Full title A cross-sectional analysis of syncytiotrophoblast membrane extracellular vesicles-derived transcriptomic biomarkers in early-onset preeclampsia. All authors Awoyemi T, Zhang W, Rahbar M, Cribbs A, Logenthiran P, Jiang S, Collett G, Cerdeira AS, Vatish M Journal Front Cardiovasc Med Abstract Preeclampsia (PE) is a pregnancy-specific hypertensive disorder affecting 2%-8% of pregnancies world (show more...)Preeclampsia (PE) is a pregnancy-specific hypertensive disorder affecting 2%-8% of pregnancies worldwide. Biomarker(s) for the disorder exists, but while these have excellent negative predictive value, their positive predictive value is poor. Extracellular vesicles released by the placenta into the maternal circulation, syncytiotrophoblast membrane extracellular vesicles (STB-EVs), have been identified as being involved in PE with the potential to act as liquid biopsies. (hide) EV-METRIC 78% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type maternal placenta perfusate Sample origin Control condition Focus vesicles small extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: Alix/ CD63/ PLAP/ CD9 non-EV: Cytochrome C Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type maternal placenta perfusate Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Sorvall TST28.39 Pelleting: speed (g) 150,000 Wash: volume per pellet (ml) 45 Wash: time (min) 120 Wash: Rotor Type Sorvall TST28.39 Wash: speed (g) 150,000 Filtration steps 0.2 or 0.22 _m Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 2250-3040 Western Blot Detected EV-associated proteins Alix/ CD63/ PLAP Not detected EV-associated proteins CD9 Not detected contaminants Cytochrome C Flow cytometry Type of Flow cytometry A BD LSRII flow cytometer (BD Biosciences) with a blue, violet, and red laser Calibration bead size 0.11/ 0.18/ 0.24/ 0.30/ 0.50/ 0.59/ 0.88/ 1.30 Proteomics database PRIDE Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR/ RNA-sequencing/ Capillary electrophoresis (e.g. Bioanalyzer) Database NCBI geo Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 479 EV concentration Yes Particle yield 1.59E11-1.78E11 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210382	3/4	Homo sapiens	maternal placenta perfusate	(d)(U)C	Awoyemi T	2023	78%
Study summary Full title A cross-sectional analysis of syncytiotrophoblast membrane extracellular vesicles-derived transcriptomic biomarkers in early-onset preeclampsia. All authors Awoyemi T, Zhang W, Rahbar M, Cribbs A, Logenthiran P, Jiang S, Collett G, Cerdeira AS, Vatish M Journal Front Cardiovasc Med Abstract Preeclampsia (PE) is a pregnancy-specific hypertensive disorder affecting 2%-8% of pregnancies world (show more...)Preeclampsia (PE) is a pregnancy-specific hypertensive disorder affecting 2%-8% of pregnancies worldwide. Biomarker(s) for the disorder exists, but while these have excellent negative predictive value, their positive predictive value is poor. Extracellular vesicles released by the placenta into the maternal circulation, syncytiotrophoblast membrane extracellular vesicles (STB-EVs), have been identified as being involved in PE with the potential to act as liquid biopsies. (hide) EV-METRIC 78% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type maternal placenta perfusate Sample origin Preeclampsia Focus vesicles medium/large extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD63/ PLAP/ CD9 non-EV: Cytochrome C Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type maternal placenta perfusate 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) 30 Pelleting: rotor type Sorvall TST28.39 Pelleting: speed (g) 10,000 Wash: volume per pellet (ml) 45 Wash: time (min) 30 Wash: Rotor Type Sorvall TST28.39 Wash: speed (g) 10,000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 10460-21650 Western Blot Detected EV-associated proteins CD63/ PLAP Not detected EV-associated proteins CD9 Not detected contaminants Cytochrome C Proteomics database PRIDE Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR/ RNA-sequencing/ Capillary electrophoresis (e.g. Bioanalyzer) Database NCBI geo Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 205.8 EV concentration Yes Particle yield 3.75E11-1E12 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210382	4/4	Homo sapiens	maternal placenta perfusate	(d)(U)C Filtration	Awoyemi T	2023	78%
Study summary Full title A cross-sectional analysis of syncytiotrophoblast membrane extracellular vesicles-derived transcriptomic biomarkers in early-onset preeclampsia. All authors Awoyemi T, Zhang W, Rahbar M, Cribbs A, Logenthiran P, Jiang S, Collett G, Cerdeira AS, Vatish M Journal Front Cardiovasc Med Abstract Preeclampsia (PE) is a pregnancy-specific hypertensive disorder affecting 2%-8% of pregnancies world (show more...)Preeclampsia (PE) is a pregnancy-specific hypertensive disorder affecting 2%-8% of pregnancies worldwide. Biomarker(s) for the disorder exists, but while these have excellent negative predictive value, their positive predictive value is poor. Extracellular vesicles released by the placenta into the maternal circulation, syncytiotrophoblast membrane extracellular vesicles (STB-EVs), have been identified as being involved in PE with the potential to act as liquid biopsies. (hide) EV-METRIC 78% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type maternal placenta perfusate Sample origin Preeclampsia Focus vesicles small extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: Alix/ CD63/ PLAP/ CD9 non-EV: Cytochrome C Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type maternal placenta perfusate Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Sorvall TST28.39 Pelleting: speed (g) 150,000 Wash: volume per pellet (ml) 45 Wash: time (min) 120 Wash: Rotor Type Sorvall TST28.39 Wash: speed (g) 150,000 Filtration steps 0.2 or 0.22 _m Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 5780-10648 Western Blot Detected EV-associated proteins Alix/ CD63/ PLAP Not detected EV-associated proteins CD9 Not detected contaminants Cytochrome C Flow cytometry Type of Flow cytometry A BD LSRII flow cytometer (BD Biosciences) with a blue, violet, and red laser Calibration bead size 0.11/ 0.18/ 0.24/ 0.30/ 0.50/ 0.59/ 0.88/ 1.30 Proteomics database PRIDE Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR/ RNA-sequencing/ Capillary electrophoresis (e.g. Bioanalyzer) Database NCBI geo Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 479 EV concentration Yes Particle yield 2.2E11-7.2E11 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210302	1/2	Homo sapiens	MKN74	(d)(U)C Filtration qEV	Poças J	2023	78%
Study summary Full title Syndecan-4 is a maestro of gastric cancer cell invasion and communication that underscores poor survival. All authors Poças J, Marques C, Gomes C, Otake AH, Pinto F, Ferreira M, Silva T, Faria-Ramos I, Matos R, Ribeiro AR, Senra E, Cavadas B, Batista S, Maia J, Macedo JA, Lima L, Afonso LP, Ferreira JA, Santos LL, Polónia A, Osório H, Belting M, Reis CA, Costa-Silva B, Magalhães A Journal Proc Natl Acad Sci U S A Abstract Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options (show more...)Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options. Here, we show that syndecan-4 (SDC4), a transmembrane proteoglycan, is highly expressed in intestinal subtype gastric tumors and that this signature associates with patient poor survival. Further, we mechanistically demonstrate that SDC4 is a master regulator of gastric cancer cell motility and invasion. We also find that SDC4 decorated with heparan sulfate is efficiently sorted in extracellular vesicles (EVs). Interestingly, SDC4 in EVs regulates gastric cancer cell-derived EV organ distribution, uptake, and functional effects in recipient cells. Specifically, we show that knockout disrupts the tropism of EVs for the common gastric cancer metastatic sites. Our findings set the basis for the molecular implications of SDC4 expression in gastric cancer cells and provide broader perspectives on the development of therapeutic strategies targeting the glycan-EV axis to limit tumor progression. (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 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 qEV Protein markers EV: Alix/ CD9/ CD63/ CD81/ HSP70/ SDCBP/ SDC4 non-EV: CytochromeC/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Albumin/ Argonaute2/ Tubulin1 Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MKN74 EV-harvesting Medium Serum free medium Cell viability (%) 88 Cell count 73000000 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) 160 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 30 Wash: time (min) 160 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Filtration steps 0.2 or 0.22 µm Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per million cells Western Blot Detected EV-associated proteins Alix/ CD9/ CD63/ CD81/ HSP70/ SDCBP/ SDC4 Not detected contaminants CytochromeC Proteomics database PRIDE Proteomics Detected contaminants Calreticulin/ GM130/ PMP70/ Prohibitin Not detected contaminants Albumin/ Argonaute2/ CytochromeC/ Tubulin1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 102.1 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.90E+08 EM EM-type Transmission-EM/ Immuno-EM EM protein SDC4 Image type Close-up, Wide-field

	EV210302	2/2	Homo sapiens	MKN74	(d)(U)C Filtration qEV	Poças J	2023	78%
Study summary Full title Syndecan-4 is a maestro of gastric cancer cell invasion and communication that underscores poor survival. All authors Poças J, Marques C, Gomes C, Otake AH, Pinto F, Ferreira M, Silva T, Faria-Ramos I, Matos R, Ribeiro AR, Senra E, Cavadas B, Batista S, Maia J, Macedo JA, Lima L, Afonso LP, Ferreira JA, Santos LL, Polónia A, Osório H, Belting M, Reis CA, Costa-Silva B, Magalhães A Journal Proc Natl Acad Sci U S A Abstract Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options (show more...)Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options. Here, we show that syndecan-4 (SDC4), a transmembrane proteoglycan, is highly expressed in intestinal subtype gastric tumors and that this signature associates with patient poor survival. Further, we mechanistically demonstrate that SDC4 is a master regulator of gastric cancer cell motility and invasion. We also find that SDC4 decorated with heparan sulfate is efficiently sorted in extracellular vesicles (EVs). Interestingly, SDC4 in EVs regulates gastric cancer cell-derived EV organ distribution, uptake, and functional effects in recipient cells. Specifically, we show that knockout disrupts the tropism of EVs for the common gastric cancer metastatic sites. Our findings set the basis for the molecular implications of SDC4 expression in gastric cancer cells and provide broader perspectives on the development of therapeutic strategies targeting the glycan-EV axis to limit tumor progression. (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 SDC4 KO Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration qEV Protein markers EV: Alix/ CD9/ CD81/ HSP70/ SDCBP non-EV: CytochromeC/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Albumin/ Argonaute2/ Tubulin1 Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MKN74 EV-harvesting Medium Serum free medium Cell viability (%) 92 Cell count 74000000 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) 160 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 30 Wash: time (min) 160 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Filtration steps 0.2 or 0.22 µm Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per million cells Western Blot Detected EV-associated proteins Alix/ CD9/ CD81/ HSP70/ SDCBP Not detected contaminants CytochromeC/ Tubulin1 Proteomics database PRIDE Proteomics Detected contaminants Calreticulin/ GM130/ PMP70/ Prohibitin Not detected contaminants Albumin/ Argonaute2/ CytochromeC/ Tubulin1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 86 EV concentration Yes Particle yield particles per milliliter of starting sample: 6.75E+08 EM EM-type Transmission-EM/ Immuno-EM EM protein SDC4 Image type Close-up, Wide-field

	EV230981	6/6	Mus musculus	Blood plasma	(d)(U)C DG qEVoriginal/70nm	André-Grégoire G	2023	75%
Study summary Full title Isolating plasma extracellular vesicles from mouse blood using size-exclusion chromatography, density gradient, and ultracentrifugation. All authors André-Grégoire G, Roux Q, Gavard J Journal STAR Protoc Abstract Circulating extracellular vesicles (EVs) could serve for the surveillance of diverse pathological co (show more...)Circulating extracellular vesicles (EVs) could serve for the surveillance of diverse pathological conditions. We present a protocol for enriching and isolating plasma EVs from mouse blood. We describe steps for employing ultracentrifugation, size-exclusion chromatography, and density gradients, required for further quantitative and qualitative analysis. We detail the procedure for retrieving optimal volume of blood while preserving its integrity and avoiding hemolysis. We also describe the preparation of EVs from this complex fluid containing soluble proteins, aggregates, and lipoprotein particles. For complete details on the use and execution of this protocol, please refer to André-Grégoire et al. (2022).. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin bearing human GSC-derived orthotopic tumour Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient qEVoriginal/70nm Protein markers EV: CD9/ Alix/ HSP70 non-EV: ApoB Proteomics no EV density (g/ml) 1.085-1.11 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100,000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 11 Pelleting: speed (g) 100,000 Commercial kit qEVoriginal/70nm Other Name other separation method qEVoriginal/70nm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9 Not detected EV-associated proteins Alix/ HSP70 Not detected contaminants ApoB Characterization: Lipid analysis No Characterization: Particle analysis Other particle analysis name(1) Interferometric light microscopy Report type Median Report size 170 EV-concentration Yes Particle yield as number of particles per milliliter of starting sample: 6.66E8

	EV230588	1/2	Homo sapiens	human umbilical cord mesenchymal stromal cells	(d)(U)C Filtration SEC (non-commercial) Tangential flow filtration	Forteza-Genestra MA	2023	75%
Study summary Full title Comparative effect of platelet- and mesenchymal stromal cell-derived extracellular vesicles on human cartilage explants using an ex vivo inflammatory osteoarthritis model. All authors Forteza-Genestra MA, Antich-Rosselló M, Ramis-Munar G, Calvo J, Gayà A, Monjo M, Ramis JM Journal Bone Joint Res Abstract Extracellular vesicles (EVs) are nanoparticles secreted by all cells, enriched in proteins, lipids, (show more...)Extracellular vesicles (EVs) are nanoparticles secreted by all cells, enriched in proteins, lipids, and nucleic acids related to cell-to-cell communication and vital components of cell-based therapies. Mesenchymal stromal cell (MSC)-derived EVs have been studied as an alternative for osteoarthritis (OA) treatment. However, their clinical translation is hindered by industrial and regulatory challenges. In contrast, platelet-derived EVs might reach clinics faster since platelet concentrates, such as platelet lysates (PL), are already used in therapeutics. Hence, we aimed to test the therapeutic potential of PL-derived extracellular vesicles (pEVs) as a new treatment for OA, which is a degenerative joint disease of articular cartilage and does not have any curative or regenerative treatment, by comparing its effects to those of human umbilical cord MSC-derived EVs (cEVs) on an ex vivo OA-induced model using human cartilage explants. (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 extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Size-exclusion chromatography (non-commercial) Tangential flow filtration Protein markers EV: Alix/ CD9/ CD63/ CD81/ HSC70 non-EV: CytC/ ApoA1 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells human umbilical cord mesenchymal stromal cells EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.2 or 0.22 ?m Size-exclusion chromatography Total column volume (mL) 120 Sample volume/column (mL) 5 Resin type Sephacryl S-400 HR Other Name other separation method Tangential flow filtration Characterization: Protein analysis Protein Concentration Method Absorbance at 280 nm Western Blot Detected EV-associated proteins Alix/ CD9/ CD63/ CD81 Not detected EV-associated proteins HSC70 Not detected contaminants CytC/ ApoA1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230588	2/2	Homo sapiens	Platelet lysate	(d)(U)C Filtration SEC (non-commercial)	Forteza-Genestra MA	2023	75%
Study summary Full title Comparative effect of platelet- and mesenchymal stromal cell-derived extracellular vesicles on human cartilage explants using an ex vivo inflammatory osteoarthritis model. All authors Forteza-Genestra MA, Antich-Rosselló M, Ramis-Munar G, Calvo J, Gayà A, Monjo M, Ramis JM Journal Bone Joint Res Abstract Extracellular vesicles (EVs) are nanoparticles secreted by all cells, enriched in proteins, lipids, (show more...)Extracellular vesicles (EVs) are nanoparticles secreted by all cells, enriched in proteins, lipids, and nucleic acids related to cell-to-cell communication and vital components of cell-based therapies. Mesenchymal stromal cell (MSC)-derived EVs have been studied as an alternative for osteoarthritis (OA) treatment. However, their clinical translation is hindered by industrial and regulatory challenges. In contrast, platelet-derived EVs might reach clinics faster since platelet concentrates, such as platelet lysates (PL), are already used in therapeutics. Hence, we aimed to test the therapeutic potential of PL-derived extracellular vesicles (pEVs) as a new treatment for OA, which is a degenerative joint disease of articular cartilage and does not have any curative or regenerative treatment, by comparing its effects to those of human umbilical cord MSC-derived EVs (cEVs) on an ex vivo OA-induced model using human cartilage explants. (hide) EV-METRIC 75% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Platelet lysate Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD63/ CD81/ HSC70 non-EV: CytC/ ApoA1 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Platelet lysate Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps Larger than 0.45 ?m Size-exclusion chromatography Total column volume (mL) 120 Sample volume/column (mL) 5 Resin type Sephacryl S-400 HR Characterization: Protein analysis Protein Concentration Method Absorbance at 280 nm Western Blot Detected EV-associated proteins CD9/ CD63 Not detected EV-associated proteins CD81/ HSC70 Not detected contaminants CytC/ ApoA1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 121 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230370	7/8	Sus scrofa	Primary retinal pigmented epithelial cells	DC DG (d)(U)C UF	Hernandez, Belinda J.	2023	75%
Study summary Full title Polarized desmosome and hemidesmosome shedding via small extracellular vesicles is an early indicator of outer blood-retina barrier dysfunction All authors Belinda J. Hernandez, Nikolai P. Skiba, Karolina Plössl, Madison Strain, Yutao Liu, Daniel Grigsby, Una Kelly, Martha A. Cady, Vikram Manocha, Arvydas Maminishkis, TeddiJo Watkins, Sheldon S. Miller, Allison Ashley-Koch, W. Daniel Stamer, Bernhard H. F. Weber, Catherine Bowes Rickman, Mikael Klingeborn Journal J Extracell Biol Abstract The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photorec (show more...)The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles (EVs) from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of small EVs (sEVs) including exosomes, that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral sEVs from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal-side sub-RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of sEV release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in sEV content, including basal-side specific desmosome and hemidesmosome shedding via sEVs. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases (e.g., AMD). (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 small extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density cushion Density gradient (Differential) (ultra)centrifugation Ultrafiltration Protein markers EV: Syntenin-1/ ANXA2/ ITGB1 non-EV: Calreticulin/ Albumin/ Lamins/ Caspases Proteomics yes EV density (g/ml) 1.07-1.11 Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Sus scrofa Sample Type Cell culture supernatant EV-producing cells Primary retinal pigmented epithelial cells EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 99 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 3 Orientation Bottom-up Speed (g) 200,000 Duration (min) 240 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: speed (g) 100,000 Density cushion Density medium Iodixanol Sample volume 36 Cushion volume 2 Density of the cushion 60% Centrifugation time 180 Centrifugation speed 100,000 Characterization: Protein analysis Protein Concentration Method Pierce 660 nm assay Western Blot Detected EV-associated proteins Syntenin-1/ ANXA2/ ITGB1/ KRT10 Not detected contaminants Calreticulin Detected contaminants Albumin Not detected contaminants Lamins/ Caspases Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 125.3 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.20E+06

	EV230370	8/8	Sus scrofa	Primary retinal pigmented epithelial cells	DC DG (d)(U)C UF	Hernandez, Belinda J.	2023	75%
Study summary Full title Polarized desmosome and hemidesmosome shedding via small extracellular vesicles is an early indicator of outer blood-retina barrier dysfunction All authors Belinda J. Hernandez, Nikolai P. Skiba, Karolina Plössl, Madison Strain, Yutao Liu, Daniel Grigsby, Una Kelly, Martha A. Cady, Vikram Manocha, Arvydas Maminishkis, TeddiJo Watkins, Sheldon S. Miller, Allison Ashley-Koch, W. Daniel Stamer, Bernhard H. F. Weber, Catherine Bowes Rickman, Mikael Klingeborn Journal J Extracell Biol Abstract The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photorec (show more...)The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles (EVs) from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of small EVs (sEVs) including exosomes, that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral sEVs from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal-side sub-RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of sEV release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in sEV content, including basal-side specific desmosome and hemidesmosome shedding via sEVs. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases (e.g., AMD). (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 0.2mM H2O2 Focus vesicles small extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density cushion Density gradient (Differential) (ultra)centrifugation Ultrafiltration Protein markers EV: Syntenin-1/ ANXA2/ ITGB1 non-EV: Calreticulin/ Albumin/ Lamins/ Caspases Proteomics yes EV density (g/ml) 1.07-1.11 Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Sus scrofa Sample Type Cell culture supernatant EV-producing cells Primary retinal pigmented epithelial cells EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 99 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 3 Orientation Bottom-up Speed (g) 200,000 Duration (min) 240 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: speed (g) 100,000 Density cushion Density medium Iodixanol Sample volume 36 Cushion volume 2 Density of the cushion 60% Centrifugation time 180 Centrifugation speed 100,000 Characterization: Protein analysis Protein Concentration Method Pierce 660 nm assay Western Blot Detected EV-associated proteins Syntenin-1/ ANXA2/ ITGB1/ KRT10 Not detected contaminants Calreticulin Detected contaminants Albumin Not detected contaminants Lamins/ Caspases Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 143.2 EV concentration Yes Particle yield particles per milliliter of starting sample: 3.80E+06

	EV230055	3/1	Homo sapiens	Brain tissues	(d)(U)C Filtration qEV UF	Yiyao Huang	2023	75%
Study summary Full title Toward a human brain extracellular vesicle atlas: Characteristics of extracellular vesicles from different brain regions, including small RNA and protein profiles All authors Yiyao Huang, Tanina Arab, Ashley E. Russell, Emily R. Mallick, Rajini Nagaraj, Evan Gizzie, Javier Redding-Ochoa, Juan C. Troncoso, Olga Pletnikova, Andrey Turchinovich, David A. Routenberg, Kenneth W. Witwer Journal Biochem Pharmacol Abstract Extracellular vesicles (EVs) are released from different cell types in the central nervous system (C (show more...)Extracellular vesicles (EVs) are released from different cell types in the central nervous system (CNS) and play roles in regulating physiological and pathological functions. Although brain-derived EVs (bdEVs) have been successfully collected from brain tissue, there is not yet a “bdEV Atlas” of EVs from different brain regions. To address this gap, we separated EVs from eight anatomical brain regions of a single individual and subsequently characterized them by count, size, morphology, and protein and RNA content. The greatest particle yield was from cerebellum, while the fewest particles were recovered from the orbitofrontal, postcentral gyrus, and thalamus regions. EV surface phenotyping indicated that CD81 and CD9 were more abundant than CD63 in all regions. Cell-enriched surface markers varied between brain regions. For example, putative neuronal markers NCAM, CD271, and NRCAM were more abundant in medulla, cerebellum, and occipital regions, respectively. These findings, while restricted to tissues from a single individual, suggest that additional studies are warranted to provide more insight into the links between EV heterogeneity and function in the CNS. (hide) EV-METRIC 75% (83rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Brain tissues Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration qEV Ultrafiltration Protein markers EV: Alix/ CD9/ CD63/ CD81/ HCAM/CD44/ CD15/ HLA-DR/DP/DQ/ GD2/ NCAM/ TSPO/ CD36/ CD38/ CD90/Thy1/ CD146/MCAM/ CD29/ CD166/hALCAM/ CD64/ CD307d/ TMEM119/ GD1a/ CD31/PECAM/ CD271/LNGFR/ CD24/ CD40/ CD163/ GJA1/ NRCAM non-EV: Calreticulin Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Brain tissues Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type TH-641 Pelleting: speed (g) 100000 Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Alix/ CD9/ CD63 Not detected contaminants Calreticulin Detected EV-associated proteins CD9/ CD63/ CD81 Detected EV-associated proteins CD9/ CD63/ CD81/ HCAM/CD44/ CD15/ HLA-DR/DP/DQ/ GD2/ NCAM/ TSPO/ CD36/ CD38/ CD90/Thy1/ CD146/MCAM/ CD29/ CD166/hALCAM/ CD64/ CD307d/ TMEM119/ GD1a/ CD31/PECAM/ CD271/LNGFR/ CD24/ CD40/ CD163/ GJA1/ Characterization: RNA analysis RNA analysis Type RNA-sequencing Database GEO Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type Flow Nanoanalyzer (NanoFCM) Hardware adjustment Compared with traditional flow cytometry, a smaller flow channel reduces background signal, and lower system pressure increases dwell time of particles for enhanced signal integration. Calibration bead size 68/ 91/ 113/ 151/ 200 Report type Size range/distribution Reported size (nm) 42-137 EV concentration Yes Particle yield 2.17-8.95E08 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 40-500

	EV230044	1/2	Homo sapiens	Blood plasma	SEC (non-commercial) UF	Herrero-Lorenzo, Marina	2023	75%
Study summary Full title Small RNAs in plasma extracellular vesicles define biomarkers of premanifest changes in Huntington’s disease All authors Marina Herrero-Lorenzo, Jesús Pérez-Pérez, Georgia Escaramís, Saül Martínez-Horta, Rocío Pérez-González, Elisa Rivas-Asensio, Jaime Kulisevsky, Ana Gámez-Valero, Eulàlia Martí Journal bioRxiv Abstract Despite the advances in the understanding of Huntington’s disease (HD), there is the need for mole (show more...)Despite the advances in the understanding of Huntington’s disease (HD), there is the need for molecular biomarkers to categorize mutation-carriers during the preclinical stage of the disease preceding the functional decline. Small RNAs (sRNAs) are a promising source of biomarkers since their expression levels are highly sensitive to pathobiological processes. Here, using an optimized method for plasma extracellular vesicles (EVs) purification and an exhaustive analysis pipeline of sRNA sequencing data, we show that EV-sRNAs are early downregulated in mutation-carriers, and that this deregulation is associated with premanifest cognitive performance. Seven candidate sRNAs (tRF-Glu-CTC, tRF-Gly-GCC, miR-451a, miR-21-5p, miR-26a-5p, miR-27a-3p, and let7a-5p) were validated in additional subjects, showing a significant diagnostic accuracy at premanifest stages. Of these, miR – 21-5p was significantly decreased over time in a longitudinal study; and miR-21-5p and miR-26a-5p levels correlated with cognitive changes in the premanifest cohort. In summary, the present results suggest that deregulated plasma EV-sRNAs define an early biosignature in mutation carriers with specific species sensing the progression and cognitive changes occurring at the premanifest stage. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Size-exclusion chromatography (non-commercial) Ultrafiltration Protein markers EV: Alix/ CD9/ Flotillin-1/ TSG101/ Syntenin/ CD63/ CD81 non-EV: Albumin/ COX4/ 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) 100 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 20 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per mililiter of pooled fractions Western Blot Detected EV-associated proteins Alix/ CD9/ Flotillin-1/ TSG101/ Syntenin Not detected contaminants Albumin/ COX4/ Calnexin Flow cytometry specific beads Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ RNA-sequencing Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 146 EV concentration Yes Particle yield number of particles per 2mL of starting sample: 8.330E09 EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 111

	EV230044	2/2	Homo sapiens	Blood plasma	SEC (non-commercial) UF	Herrero-Lorenzo, Marina	2023	75%
Study summary Full title Small RNAs in plasma extracellular vesicles define biomarkers of premanifest changes in Huntington’s disease All authors Marina Herrero-Lorenzo, Jesús Pérez-Pérez, Georgia Escaramís, Saül Martínez-Horta, Rocío Pérez-González, Elisa Rivas-Asensio, Jaime Kulisevsky, Ana Gámez-Valero, Eulàlia Martí Journal bioRxiv Abstract Despite the advances in the understanding of Huntington’s disease (HD), there is the need for mole (show more...)Despite the advances in the understanding of Huntington’s disease (HD), there is the need for molecular biomarkers to categorize mutation-carriers during the preclinical stage of the disease preceding the functional decline. Small RNAs (sRNAs) are a promising source of biomarkers since their expression levels are highly sensitive to pathobiological processes. Here, using an optimized method for plasma extracellular vesicles (EVs) purification and an exhaustive analysis pipeline of sRNA sequencing data, we show that EV-sRNAs are early downregulated in mutation-carriers, and that this deregulation is associated with premanifest cognitive performance. Seven candidate sRNAs (tRF-Glu-CTC, tRF-Gly-GCC, miR-451a, miR-21-5p, miR-26a-5p, miR-27a-3p, and let7a-5p) were validated in additional subjects, showing a significant diagnostic accuracy at premanifest stages. Of these, miR – 21-5p was significantly decreased over time in a longitudinal study; and miR-21-5p and miR-26a-5p levels correlated with cognitive changes in the premanifest cohort. In summary, the present results suggest that deregulated plasma EV-sRNAs define an early biosignature in mutation carriers with specific species sensing the progression and cognitive changes occurring at the premanifest stage. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Huntington's disease Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Size-exclusion chromatography (non-commercial) Ultrafiltration Protein markers EV: Alix/ CD9/ Flotillin-1/ TSG101/ Syntenin/ CD63/ CD81 non-EV: Albumin/ COX4/ 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) 100 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 20 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per mililiter of pooled fractions Western Blot Detected EV-associated proteins Alix/ CD9/ Flotillin-1/ TSG101/ Syntenin Not detected contaminants Albumin/ COX4/ Calnexin Flow cytometry specific beads Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ RNA-sequencing Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 80-300 EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV230013	1/3	Homo sapiens	Blood plasma	SEC (non-commercial)	Darragh IAJ	2023	75%
Study summary Full title The separation and identification of circulating small extracellular vesicles from endurance-trained, strength-trained and recreationally active men. All authors Darragh IAJ, McNamee N, Daly R, Pacheco SM, O'Driscoll L, Egan B Journal J Physiol Abstract Small extracellular vesicles (EV) are membrane-encapsulated particles that carry bioactive cargoes, (show more...)Small extracellular vesicles (EV) are membrane-encapsulated particles that carry bioactive cargoes, are released by all cell types and are present in all human biofluids. Changes in EV profiles and abundance occur in response to acute exercise, but this study investigated whether individuals with divergent histories of exercise training (recreationally active controls - CON/ endurance-trained - END/ strength-trained - STR) presented with varied abundances of small EVs in resting samples and whether the abundance of small EVs differed within each group across two measurement days. Participants (n = 38, all male/ CON n = 12, END n = 13, STR n = 13) arrived at the lab on two separate occasions in a rested, overnight fasted state, with standardisation of time of day of sampling, recent dietary intake, time since last meal and time since last exercise training session (∼40 h). Whole blood samples were collected and separated into plasma from which small EVs were separated using size exclusion chromatography and identified in accordance with the Minimal Information For Studies of Extracellular Vesicles (MISEV) guidelines. No differences in the abundance of small EVs were observed within or between groups across multiple methods of small EV identification (nanoparticle tracking analysis, flow cytometry, immunoblot of specific EV markers). Targeted metabolomics of the small EV preparations identified 96 metabolites that were associated with the structure and function of small EVs, with no statistically significant differences in concentrations observed across groups. The results of the current study suggest that the abundance and metabolomic profile of small EVs derived from men with divergent histories of exercise training are similar to those in resting blood samples. KEY POINTS: Extracellular vesicles (EV) are membrane-encapsulated particles that are present in circulation and carry bioactive materials as 'cargo'. The abundance and profile of small EVs are responsive to acute exercise, but little is known about the relationship between small EVs and exercise training. This study examined the abundance, and a targeted metabolomic profile, of small EVs separated from the blood of endurance athletes, strength athletes and recreationally active controls at rest (∼40 h after the most recent exercise session) on two separate but identical lab visits. No differences were observed in the abundance or metabolomic profile of small EV preparations between the groups or between the lab visits within each group. Further research should determine whether the bioactive cargoes (e.g. RNA, protein and additional metabolites) carried within EVs are altered in individuals with divergent histories of exercise training or in response to exercise training interventions. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Size-exclusion chromatography (non-commercial) Protein markers EV: CD63/ Flotillin-1/ Alix/ CD9/ TSG101 non-EV: Albumin Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Size-exclusion chromatography Sample volume/column (mL) 1 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ Flotillin-1 Not detected EV-associated proteins Alix/ CD9/ TSG101 Detected contaminants Albumin Flow cytometry aspecific beads Detected EV-associated proteins CD63 Flow cytometry specific beads Selected surface protein(s) CD63 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 135 EV concentration Yes Particle yield particles per milliliter of starting sample: 75084686.28/mL Particle analysis: flow cytometry Flow cytometer type ImageStream X MK II Hardware adjustment 60X magnification and low flow rate Calibration bead size 6 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.00E+06 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230013	2/3	Homo sapiens	Blood plasma	SEC (non-commercial)	Darragh IAJ	2023	75%
Study summary Full title The separation and identification of circulating small extracellular vesicles from endurance-trained, strength-trained and recreationally active men. All authors Darragh IAJ, McNamee N, Daly R, Pacheco SM, O'Driscoll L, Egan B Journal J Physiol Abstract Small extracellular vesicles (EV) are membrane-encapsulated particles that carry bioactive cargoes, (show more...)Small extracellular vesicles (EV) are membrane-encapsulated particles that carry bioactive cargoes, are released by all cell types and are present in all human biofluids. Changes in EV profiles and abundance occur in response to acute exercise, but this study investigated whether individuals with divergent histories of exercise training (recreationally active controls - CON/ endurance-trained - END/ strength-trained - STR) presented with varied abundances of small EVs in resting samples and whether the abundance of small EVs differed within each group across two measurement days. Participants (n = 38, all male/ CON n = 12, END n = 13, STR n = 13) arrived at the lab on two separate occasions in a rested, overnight fasted state, with standardisation of time of day of sampling, recent dietary intake, time since last meal and time since last exercise training session (∼40 h). Whole blood samples were collected and separated into plasma from which small EVs were separated using size exclusion chromatography and identified in accordance with the Minimal Information For Studies of Extracellular Vesicles (MISEV) guidelines. No differences in the abundance of small EVs were observed within or between groups across multiple methods of small EV identification (nanoparticle tracking analysis, flow cytometry, immunoblot of specific EV markers). Targeted metabolomics of the small EV preparations identified 96 metabolites that were associated with the structure and function of small EVs, with no statistically significant differences in concentrations observed across groups. The results of the current study suggest that the abundance and metabolomic profile of small EVs derived from men with divergent histories of exercise training are similar to those in resting blood samples. KEY POINTS: Extracellular vesicles (EV) are membrane-encapsulated particles that are present in circulation and carry bioactive materials as 'cargo'. The abundance and profile of small EVs are responsive to acute exercise, but little is known about the relationship between small EVs and exercise training. This study examined the abundance, and a targeted metabolomic profile, of small EVs separated from the blood of endurance athletes, strength athletes and recreationally active controls at rest (∼40 h after the most recent exercise session) on two separate but identical lab visits. No differences were observed in the abundance or metabolomic profile of small EV preparations between the groups or between the lab visits within each group. Further research should determine whether the bioactive cargoes (e.g. RNA, protein and additional metabolites) carried within EVs are altered in individuals with divergent histories of exercise training or in response to exercise training interventions. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Elite Level Endurance Athletes 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) Protein markers EV: CD63/ Flotillin-1/ Alix/ CD9/ TSG101 non-EV: Albumin Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Size-exclusion chromatography Sample volume/column (mL) 1 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ Flotillin-1 Not detected EV-associated proteins Alix/ CD9/ TSG101 Detected contaminants Albumin Flow cytometry aspecific beads Detected EV-associated proteins CD63 Flow cytometry specific beads Selected surface protein(s) CD63 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 131 EV concentration Yes Particle yield particles per milliliter of starting sample: 25937424601/mL Particle analysis: flow cytometry Flow cytometer type ImageStream X MK II Hardware adjustment 60X magnification and low flow rate Calibration bead size 6 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.50E+06 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230013	3/3	Homo sapiens	Blood plasma	SEC (non-commercial)	Darragh IAJ	2023	75%
Study summary Full title The separation and identification of circulating small extracellular vesicles from endurance-trained, strength-trained and recreationally active men. All authors Darragh IAJ, McNamee N, Daly R, Pacheco SM, O'Driscoll L, Egan B Journal J Physiol Abstract Small extracellular vesicles (EV) are membrane-encapsulated particles that carry bioactive cargoes, (show more...)Small extracellular vesicles (EV) are membrane-encapsulated particles that carry bioactive cargoes, are released by all cell types and are present in all human biofluids. Changes in EV profiles and abundance occur in response to acute exercise, but this study investigated whether individuals with divergent histories of exercise training (recreationally active controls - CON/ endurance-trained - END/ strength-trained - STR) presented with varied abundances of small EVs in resting samples and whether the abundance of small EVs differed within each group across two measurement days. Participants (n = 38, all male/ CON n = 12, END n = 13, STR n = 13) arrived at the lab on two separate occasions in a rested, overnight fasted state, with standardisation of time of day of sampling, recent dietary intake, time since last meal and time since last exercise training session (∼40 h). Whole blood samples were collected and separated into plasma from which small EVs were separated using size exclusion chromatography and identified in accordance with the Minimal Information For Studies of Extracellular Vesicles (MISEV) guidelines. No differences in the abundance of small EVs were observed within or between groups across multiple methods of small EV identification (nanoparticle tracking analysis, flow cytometry, immunoblot of specific EV markers). Targeted metabolomics of the small EV preparations identified 96 metabolites that were associated with the structure and function of small EVs, with no statistically significant differences in concentrations observed across groups. The results of the current study suggest that the abundance and metabolomic profile of small EVs derived from men with divergent histories of exercise training are similar to those in resting blood samples. KEY POINTS: Extracellular vesicles (EV) are membrane-encapsulated particles that are present in circulation and carry bioactive materials as 'cargo'. The abundance and profile of small EVs are responsive to acute exercise, but little is known about the relationship between small EVs and exercise training. This study examined the abundance, and a targeted metabolomic profile, of small EVs separated from the blood of endurance athletes, strength athletes and recreationally active controls at rest (∼40 h after the most recent exercise session) on two separate but identical lab visits. No differences were observed in the abundance or metabolomic profile of small EV preparations between the groups or between the lab visits within each group. Further research should determine whether the bioactive cargoes (e.g. RNA, protein and additional metabolites) carried within EVs are altered in individuals with divergent histories of exercise training or in response to exercise training interventions. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Elite Level Strength Athletes 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) Protein markers EV: CD63/ Flotillin-1/ Alix/ CD9/ TSG101 non-EV: Albumin Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Size-exclusion chromatography Sample volume/column (mL) 1 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ Flotillin-1 Not detected EV-associated proteins Alix/ CD9/ TSG101 Detected contaminants Albumin Flow cytometry aspecific beads Detected EV-associated proteins CD63 Flow cytometry specific beads Selected surface protein(s) CD63 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 131 EV concentration Yes Particle yield particles per milliliter of starting sample: 33945673899/mL Particle analysis: flow cytometry Flow cytometer type ImageStream X MK II Hardware adjustment 60X magnification and low flow rate Calibration bead size 6 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.50E+06 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220321	8/8	Homo sapiens	Blood plasma	DG UF SEC (non-commercial)	Kashkanova AD	2023	75%
Study summary Full title Label-free discrimination of extracellular vesicles from large lipoproteins. All authors Kashkanova AD, Blessing M, Reischke M, Baur JO, Baur AS, Sandoghdar V, Van Deun J Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are increasingly gaining interest as biomarkers and therapeutics. Accur (show more...)Extracellular vesicles (EVs) are increasingly gaining interest as biomarkers and therapeutics. Accurate sizing and quantification of EVs remain problematic, given their nanometre size range and small scattering cross-sections. This is compounded by the fact that common EV isolation methods result in co-isolation of particles with comparable features. Especially in blood plasma, similarly-sized lipoproteins outnumber EVs to a great extent. Recently, interferometric nanoparticle tracking analysis (iNTA) was introduced as a particle analysis method that enables determining the size and refractive index of nanoparticles with high sensitivity and precision. In this work, we apply iNTA to differentiate between EVs and lipoproteins, and compare its performance to conventional nanoparticle tracking analysis (NTA). We show that iNTA can accurately quantify EVs in artificial EV-lipoprotein mixtures and in plasma-derived EV samples of varying complexity. Conventional NTA could not report on EV numbers, as it was not able to distinguish EVs from lipoproteins. iNTA has the potential to become a new standard for label-free EV characterization in suspension. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Melanoma Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD63/ CD81 non-EV: ApoB Proteomics no EV density (g/ml) 1.1-1.2 Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 11.5 Sample volume (mL) 0.5 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-4B Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63/ CD81 ELISA Characterization: Lipid analysis No Characterization: Particle analysis Other particle analysis name(1) interferometric nanoparticle tracking analysis EV-concentration Yes Particle yield No

	EV210141	4/5	Homo sapiens	human umbilical vein endothelial cells	Ultrafiltratrion (d)(U)C DG	Zhao F	2023	75%
Study summary Full title Extracellular vesicles from Zika virus-infected cells display viral E protein that binds ZIKV-neutralizing antibodies to prevent infection enhancement. All authors Zhao F, Xu Y, Liu N, Lv D, Chen Y, Liu Z, Jin X, Xiao M, Lavillette D, Zhong J, Bartenschlager R, Long G Journal EMBO J Abstract Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some pa (show more...)Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some parts of the world. Although ZIKV infection is predominantly asymptomatic or associated with mild symptoms, it can lead to neurological complications. ZIKV infection can also cause antibody-dependent enhancement (ADE) of infection with similar viruses, warranting further studies of virion assembly and the function of envelope (E) protein-specific antibodies. Although extracellular vesicles (EVs) from flavivirus-infected cells have been reported to transmit infection, this interpretation is challenged by difficulties in separating EVs from flavivirions due to their similar biochemical composition and biophysical properties. In the present study, a rigorous EV-virion separation method combining sequential ultracentrifugation and affinity capture was developed to study EVs from ZIKV-infected cells. We find that these EVs do not transmit infection, but EVs display abundant E proteins which have an antigenic landscape similar to that of virions carrying E. ZIKV E-coated EVs attenuate antibody-dependent enhancement mediated by ZIKV E-specific and DENV-cross-reactive antibodies in both cell culture and mouse models. We thus report an alternative route for Flavivirus E protein secretion. These results suggest that modulation of E protein release via virions and EVs may present a new approach to regulating flavivirus-host interactions. (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 ZIKV infected cells 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 Ultrafiltratrion (Differential) (ultra)centrifugation Density gradient Protein markers EV: TSG101/ CD63/ CD81/ Alix/ HSP70/ CD9 non-EV: Capsid/ E Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells human umbilical vein endothelial cells EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell count 2,00E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 0% Highest density fraction 80% Sample volume (mL) 1 Orientation Bottom-up Rotor type P55ST Speed (g) 250000 Duration (min) 1080 Fraction volume (mL) 0,3 Ultra filtration Cut-off size (kDa) 100 kDa Membrane type Regenerated cellulose Other Name other separation method Ultrafiltratrion Characterization: Protein analysis Western Blot Detected EV-associated proteins CD9/ CD63/ TSG101/ HSP70/ Syntenin Detected contaminants Capsid/ E Characterization: RNA analysis RNA analysis Type RT(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No

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