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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	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 Antibody details provided? No 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 Antibody details provided? No 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 Antibody details provided? No 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

	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 Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes 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 Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes 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 Antibody details provided? No 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 Antibody details provided? No 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% (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 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

	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 Antibody details provided? No 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 Antibody details provided? No 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 Antibody details provided? No 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 Antibody details provided? No 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% (98th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 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 Antibody details provided? No 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% (98th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 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 Antibody details provided? No 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% (98th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 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 Antibody details provided? No 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

	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% (98th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 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 Antibody details provided? No 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% (98th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 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 Antibody details provided? No 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

	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 Antibody details provided? No 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 Antibody details provided? No 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 Antibody details provided? No 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 Antibody details provided? No 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 Antibody details provided? No 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

	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 Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin-1 Not detected EV-associated proteins Alix/ CD9/ TSG101 Detected contaminants Albumin Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD63 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? Yes 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 Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin-1 Not detected EV-associated proteins Alix/ CD9/ TSG101 Detected contaminants Albumin Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD63 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? Yes 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 Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin-1 Not detected EV-associated proteins Alix/ CD9/ TSG101 Detected contaminants Albumin Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD63 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? Yes 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 Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81 ELISA Antibody details provided? No 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 Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes 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

	EV220363	4/4	Rattus norvegicus	PC12	(d)(U)C DG	Tedford E	2023	71%
Study summary Full title Infection-induced extracellular vesicles evoke neuronal transcriptional and epigenetic changes. All authors Tedford E, Badya NB, Laing C, Asaoka N, Kaneko S, Filippi BM, McConkey GA Journal Sci Rep Abstract Infection with the protozoan Toxoplasma gondii induces changes in neurotransmission, neuroinflammati (show more...)Infection with the protozoan Toxoplasma gondii induces changes in neurotransmission, neuroinflammation, and behavior, yet it remains elusive how these changes come about. In this study we investigated how norepinephrine levels are altered by infection. TINEV (Toxoplasma-induced neuronal extracellular vesicles) isolated from infected noradrenergic cells down-regulated dopamine ß-hydroxylase (DBH) gene expression in human and rodent cells. Here we report that intracerebral injection of TINEVs into the brain is sufficient to induce DBH down-regulation and distrupt catecholaminergic signalling. Further, TINEV treatment induced hypermethylation upstream of the DBH gene. An antisense lncRNA to DBH was found in purified TINEV preparations. Paracrine signalling to induce transcriptional gene silencing and DNA methylation may be a common mode to regulate neurologic function. (hide) EV-METRIC 71% (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 Toxoplasma gondii Prugniaud-infected Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: CD63/ CD81/ Flotillin-1/ TSG101/ EpCAM/ Alix non-EV: GM130 Proteomics no EV density (g/ml) 1.16 Show all info Study aim Function Sample Species Rattus norvegicus Sample Type Cell culture supernatant EV-producing cells PC12 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 160000 Density gradient Type Discontinuous Number of initial discontinuous layers 11 Lowest density fraction 10% Highest density fraction 70% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 70000 Duration (min) 960 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: speed (g) 160000 Pelleting: adjusted k-factor 1.132 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Other 1 Dot Blot SBI Detected EV-associated proteins CD63/ CD81/ Flotillin1/ TSG101/ EpCAM Not detected EV-associated proteins Alix Detected contaminants none Not detected contaminants GM130 Characterization: RNA analysis RNA analysis Type RT-PCR 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

	EV230965	2/2	Homo sapiens	urine	(d)(U)C ExoQuick	Tao W	2023	67%
Study summary Full title A urine extracellular vesicle lncRNA classifier for high-grade prostate cancer and increased risk of progression: A multi-center study. All authors Tao W, Wang BY, Luo L, Li Q, Meng ZA, Xia TL, Deng WM, Yang M, Zhou J, Zhang X, Gao X, Li LY, He YD Journal Cell Rep Med Abstract To construct a urine extracellular vesicle long non-coding RNA (lncRNA) classifier that can detect h (show more...)To construct a urine extracellular vesicle long non-coding RNA (lncRNA) classifier that can detect high-grade prostate cancer (PCa) of grade group 2 or greater and estimate the risk of progression during active surveillance, we identify high-grade PCa-specific lncRNAs by combined analyses of cohorts from TAHSY, TCGA, and the GEO database. We develop and validate a 3-lncRNA diagnostic model (C, being made of AC015987.1, CTD-2589M5.4, RP11-363E6.3) that can detect high-grade PCa. C shows higher accuracy than prostate cancer antigen 3 (PCA3), multiparametric magnetic resonance imaging (mpMRI), and two risk calculators (Prostate Cancer Prevention Trial [PCPT]-RC 2.0 and European Randomized Study of Screening for Prostate Cancer [ERSPC]-RC) in the training cohort (n = 350), two independent cohorts (n = 232/ n = 251), and TCGA cohort (n = 499). In the prospective active surveillance cohort (n = 182), C at diagnosis remains a powerful independent predictor for overall active surveillance progression. Thus, C is a potential biomarker for high-grade PCa and can also serve as a biomarker for improved selection of candidates for active surveillance. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type urine Sample origin prostate cancer Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation ExoQuick Protein markers EV: CD9/ CD63/ CD81/ HSP70 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 5 Wash: time (min) 30 Wash: speed (g) 1500 Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81/ HSP70 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR/ RNAsequencing/ dd-PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 133 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.68E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230962	1/6	Bos bovis	bovine milk	(d)(U)C	Colella, Anna P.	2023	67%
Study summary Full title Homogenization and thermal processing reduce the concentration of extracellular vesicles in bovine milk All authors Anna P. Colella, Anuradha Prakash, John J. Miklavcic Journal Food Science and Nutrition Abstract Extracellular vesicles (EVs) in bovine milk confer beneficial physiologic effects to consumers. Indu (show more...)Extracellular vesicles (EVs) in bovine milk confer beneficial physiologic effects to consumers. Industrial processing treatments may affect the amount or bioactivity of EVs intrinsic to bovine milk. We investigated how the content and concentration of EVs were affected by homogenization and thermal processing of raw bovine milk. Raw milk was processed by homogenization, low-temperature (LT) heat, or pasteurization [high-temperature short-time (HTST) and ultra-high-temperature (UHT)] in a pilot processing facility. EVs were isolated from the raw and processed bovine milk using differential ultracentrifugation and quantified using a nanoparticle tracking analyzer. Bovine milk EVs were assessed for total miRNA and protein concentrations standardized to particle count using a fluorometric assay. There were 1.01 × 1010 (±3.30 × 109) EV particles per ml of bovine milk. All industrial processing treatments caused >60% decrease in EV concentration compared to the raw bovine milk. Homogenization and heat treatments independently and additively reduced the content of EVs in bovine milk. The averages of total miRNA/particle and total protein/particle concentrations were elevated threefold by low-temperature heat-processing treatment relative to HTST and UHT pasteurizations. The average diameter of EVs was reduced by 11%–16% by low temperature compared to raw milk (127 ± 13 nm). Homogenization and pasteurization indiscriminately reduce the EV concentration of bovine milk. Smaller EVs with higher protein content resist degradation when processing bovine milk at sub-pasteurization temperature. This new foundational knowledge may contribute to food product development on the preservation of EVs in processed dairy products, including bovine milk-based infant formulas that some newborns are dependent on for adequate growth and development. (hide) EV-METRIC 67% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type bovine milk 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/ CD63/ CD81/ Flotillin-1/ TSG101/ ANXA5/ EpCAM/ ICAM1 non-EV: GM130 Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Bos bovis Sample Type bovine milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 130000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Detected EV-associated proteins Alix/ CD63/ CD81/ Flotillin-1/ TSG101/ ANXA5/ EpCAM/ ICAM1 Not detected EV-associated proteins EpCAM Not detected contaminants GM130 Characterization: RNA analysis RNA analysis Type fluorometric for total miRNA Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 127 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.01E+10 EM EM-type Scanning-EM Image type Close-up, Wide-field

	EV230603	1/4	Bos taurus	Blood plasma	(d)(U)C qEV Filtration	Turner N	2023	67%
Study summary Full title SWATH-MS Analysis of Blood Plasma and Circulating Small Extracellular Vesicles Enables Detection of Putative Protein Biomarkers of Fertility in Young and Aged Dairy Cows. All authors Turner N, Abeysinghe P, Flay H, Meier S, Sadowski P, Mitchell MD Journal J Proteome Res Abstract The development of biomarkers of fertility could provide benefits for the genetic improvement of dai (show more...)The development of biomarkers of fertility could provide benefits for the genetic improvement of dairy cows. Circulating small extracellular vesicles (sEVs) show promise as diagnostic or prognostic markers since their cargo reflects the metabolic state of the cell of origin/ thus, they mirror the physiological status of the host. Here, we employed data-independent acquisition mass spectrometry to survey the plasma and plasma sEV proteomes of two different cohorts of Young (Peripubertal/ = 30) and Aged (Primiparous/ = 20) dairy cows () of high- and low-genetic merit of fertility and known pregnancy outcomes (ProteomeXchange data set identifier PXD042891). We established predictive models of fertility status with an area under the curve of 0.97 (sEV/ value = 3.302e-07) and 0.95 (plasma/ value = 6.405e-08). Biomarker candidates unique to high-fertility Young cattle had a sensitivity of 0.77 and specificity of 0.67 (* = 0.0287). Low-fertility biomarker candidates uniquely identified in sEVs from Young and Aged cattle had a sensitivity and specificity of 0.69 and 1.0, respectively (*** = 0.0005). Our bioinformatics pipeline enabled quantification of plasma and circulating sEV proteins associated with fertility phenotype. Further investigations are warranted to validate this research in a larger population, which may lead to improved classification of fertility status in cattle. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Low-fertility Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Filtration Protein markers EV: CD9/ CD81/ Flotillin-1/ TSG101/ GAPDH non-EV: Albumin Proteomics yes Show all info Study aim Biomarker Sample Species Bos taurus 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 50.2 Ti Pelleting: speed (g) 120,000 Filtration steps 0.2 or 0.22 ?m Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD81/ Flotillin-1/ TSG101 Not detected EV-associated proteins GAPDH Detected contaminants Albumin Proteomics database No Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 100 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230603	2/4	Bos taurus	Blood plasma	(d)(U)C qEV Filtration	Turner N	2023	67%
Study summary Full title SWATH-MS Analysis of Blood Plasma and Circulating Small Extracellular Vesicles Enables Detection of Putative Protein Biomarkers of Fertility in Young and Aged Dairy Cows. All authors Turner N, Abeysinghe P, Flay H, Meier S, Sadowski P, Mitchell MD Journal J Proteome Res Abstract The development of biomarkers of fertility could provide benefits for the genetic improvement of dai (show more...)The development of biomarkers of fertility could provide benefits for the genetic improvement of dairy cows. Circulating small extracellular vesicles (sEVs) show promise as diagnostic or prognostic markers since their cargo reflects the metabolic state of the cell of origin/ thus, they mirror the physiological status of the host. Here, we employed data-independent acquisition mass spectrometry to survey the plasma and plasma sEV proteomes of two different cohorts of Young (Peripubertal/ = 30) and Aged (Primiparous/ = 20) dairy cows () of high- and low-genetic merit of fertility and known pregnancy outcomes (ProteomeXchange data set identifier PXD042891). We established predictive models of fertility status with an area under the curve of 0.97 (sEV/ value = 3.302e-07) and 0.95 (plasma/ value = 6.405e-08). Biomarker candidates unique to high-fertility Young cattle had a sensitivity of 0.77 and specificity of 0.67 (* = 0.0287). Low-fertility biomarker candidates uniquely identified in sEVs from Young and Aged cattle had a sensitivity and specificity of 0.69 and 1.0, respectively (*** = 0.0005). Our bioinformatics pipeline enabled quantification of plasma and circulating sEV proteins associated with fertility phenotype. Further investigations are warranted to validate this research in a larger population, which may lead to improved classification of fertility status in cattle. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin High-fertility Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Filtration Protein markers EV: CD9/ CD81/ Flotillin-1/ TSG101/ GAPDH non-EV: Albumin Proteomics yes Show all info Study aim Biomarker Sample Species Bos taurus 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 50.2 Ti Pelleting: speed (g) 120,000 Filtration steps 0.2 or 0.22 ?m Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD81/ Flotillin-1/ TSG101 Not detected EV-associated proteins GAPDH Detected contaminants Albumin Proteomics database No Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 100 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230603	3/4	Bos taurus	Blood plasma	(d)(U)C qEV	Turner N	2023	67%
Study summary Full title SWATH-MS Analysis of Blood Plasma and Circulating Small Extracellular Vesicles Enables Detection of Putative Protein Biomarkers of Fertility in Young and Aged Dairy Cows. All authors Turner N, Abeysinghe P, Flay H, Meier S, Sadowski P, Mitchell MD Journal J Proteome Res Abstract The development of biomarkers of fertility could provide benefits for the genetic improvement of dai (show more...)The development of biomarkers of fertility could provide benefits for the genetic improvement of dairy cows. Circulating small extracellular vesicles (sEVs) show promise as diagnostic or prognostic markers since their cargo reflects the metabolic state of the cell of origin/ thus, they mirror the physiological status of the host. Here, we employed data-independent acquisition mass spectrometry to survey the plasma and plasma sEV proteomes of two different cohorts of Young (Peripubertal/ = 30) and Aged (Primiparous/ = 20) dairy cows () of high- and low-genetic merit of fertility and known pregnancy outcomes (ProteomeXchange data set identifier PXD042891). We established predictive models of fertility status with an area under the curve of 0.97 (sEV/ value = 3.302e-07) and 0.95 (plasma/ value = 6.405e-08). Biomarker candidates unique to high-fertility Young cattle had a sensitivity of 0.77 and specificity of 0.67 (* = 0.0287). Low-fertility biomarker candidates uniquely identified in sEVs from Young and Aged cattle had a sensitivity and specificity of 0.69 and 1.0, respectively (*** = 0.0005). Our bioinformatics pipeline enabled quantification of plasma and circulating sEV proteins associated with fertility phenotype. Further investigations are warranted to validate this research in a larger population, which may lead to improved classification of fertility status in cattle. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Low-fertility Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Protein markers EV: CD9/ CD81/ Flotillin-1/ TSG101/ GAPDH non-EV: Albumin Proteomics yes Show all info Study aim Biomarker Sample Species Bos taurus 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 70.1 Ti Pelleting: speed (g) 100,000 Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD81/ Flotillin-1/ TSG101 Not detected EV-associated proteins GAPDH Detected contaminants Albumin Proteomics database No Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 125 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230603	4/4	Bos taurus	Blood plasma	(d)(U)C qEV	Turner N	2023	67%
Study summary Full title SWATH-MS Analysis of Blood Plasma and Circulating Small Extracellular Vesicles Enables Detection of Putative Protein Biomarkers of Fertility in Young and Aged Dairy Cows. All authors Turner N, Abeysinghe P, Flay H, Meier S, Sadowski P, Mitchell MD Journal J Proteome Res Abstract The development of biomarkers of fertility could provide benefits for the genetic improvement of dai (show more...)The development of biomarkers of fertility could provide benefits for the genetic improvement of dairy cows. Circulating small extracellular vesicles (sEVs) show promise as diagnostic or prognostic markers since their cargo reflects the metabolic state of the cell of origin/ thus, they mirror the physiological status of the host. Here, we employed data-independent acquisition mass spectrometry to survey the plasma and plasma sEV proteomes of two different cohorts of Young (Peripubertal/ = 30) and Aged (Primiparous/ = 20) dairy cows () of high- and low-genetic merit of fertility and known pregnancy outcomes (ProteomeXchange data set identifier PXD042891). We established predictive models of fertility status with an area under the curve of 0.97 (sEV/ value = 3.302e-07) and 0.95 (plasma/ value = 6.405e-08). Biomarker candidates unique to high-fertility Young cattle had a sensitivity of 0.77 and specificity of 0.67 (* = 0.0287). Low-fertility biomarker candidates uniquely identified in sEVs from Young and Aged cattle had a sensitivity and specificity of 0.69 and 1.0, respectively (*** = 0.0005). Our bioinformatics pipeline enabled quantification of plasma and circulating sEV proteins associated with fertility phenotype. Further investigations are warranted to validate this research in a larger population, which may lead to improved classification of fertility status in cattle. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin High-fertility Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Protein markers EV: CD9/ CD81/ Flotillin-1/ TSG101/ GAPDH non-EV: Albumin Proteomics yes Show all info Study aim Biomarker Sample Species Bos taurus 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 70.1 Ti Pelleting: speed (g) 100,000 Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD81/ Flotillin-1/ TSG101 Not detected EV-associated proteins GAPDH Detected contaminants Albumin Proteomics database No Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 112 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230602	3/3	Homo sapiens	Blood plasma	(d)(U)C	Benayas, Beatriz	2023	67%
Study summary Full title Optimization of extracellular vesicle isolation and their separation from lipoproteins by size exclusion chromatography All authors Beatriz Benayas, Joaquín Morales, Carolina Egea, Pilar Armisén, María Yáñez-Mó Journal J Extracell Biol Abstract Interest in the use of extracellular vesicles (EVs) as biomarkers of disease is rapidly growing. How (show more...)Interest in the use of extracellular vesicles (EVs) as biomarkers of disease is rapidly growing. However, one main unsolved issue in the EV field is finding a technique able to eliminate non-EV contaminants present in biofluid samples in a one-step isolation protocol. Due to the expansion and value of size exclusion chromatography (SEC) as one of the best EV isolation methods, we have tested several agarose resins with different agarose percentages, bead sizes and crosslinking features to optimize EV isolation. For this optimization of SEC, we first employed conditioned media from a melanoma cell culture, a simpler sample in comparison to biological fluids, but which also contains abundant contaminants such as soluble protein and lipoproteins (LPPs). The distinct agaroses and the combinations of resins with different agarose percentages in the same column were tested. Soluble protein, EVs and LPPs levels from the different eluted fractions were quantitated by immunodetection or absorbance measurements. Samples were also analysed by NTA and TEM to verify the yield and the LPP contamination. Different percentages of agarose resins (2%, 4% and 6%) yielded samples with increasing LPP contamination respectively, which was not improved in the columns that combined them. Crosslinking of the agarose did not affect EV isolation yield nor the LPP contamination. In contrast, reducing the bead size greatly improved EV purity. We thus selected 4% Rapid Run Fine agarose beads as the resin that more efficiently isolated EVs with almost no contamination of other particles. Using blood plasma samples, this resin also demonstrated an improved capacity in the isolation of EVs from LPPs in comparison to the agaroses most commonly used in the field and differential ultracentrifugation. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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/ CD81 non-EV: ApoB/ ApoE Proteomics no 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 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 AH 627 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD81 Not detected contaminants ApoB/ ApoE Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 120 EV concentration Yes Particle yield particles per milliliter of starting sample: 3.00E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230374	2/13	Homo sapiens	HEK293T	(d)(U)C	Levy-Myers R	2023	67%
Study summary Full title An independent regulator of global release pathways in astrocytes generates a subtype of extracellular vesicles required for postsynaptic function. All authors Levy-Myers R, Daudelin D, Na CH, Sockanathan S Journal Sci Adv Abstract Extracellular vesicles (EVs) are heterogeneous in size, composition, and function. We show that the (show more...)Extracellular vesicles (EVs) are heterogeneous in size, composition, and function. We show that the six-transmembrane protein glycerophosphodiester phosphodiesterase 3 (GDE3) regulates actin remodeling, a global EV biogenic pathway, to release an EV subtype with distinct functions. GDE3 is necessary and sufficient for releasing EVs containing annexin A1 and GDE3 from the plasma membrane via Wiskott-Aldrich syndrome protein family member 3 (WAVE3), a major regulator of actin dynamics. GDE3 is expressed in astrocytes but not neurons, yet mice lacking GDE3 [ knockout (KO)] have decreased miniature excitatory postsynaptic current (mEPSC) amplitudes in hippocampal CA1 neurons. EVs from cultured wild-type astrocytes restore mEPSC amplitudes in KOs, while EVs from KO astrocytes or astrocytes inhibited for WAVE3 actin branching activity do not. Thus, GDE3-WAVE3 is a nonredundant astrocytic pathway that remodels actin to release a functionally distinct EV subtype, supporting the concept that independent regulation of global EV release pathways differentially regulates EV signaling within the cellular EV landscape. (hide) EV-METRIC 67% (95th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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: CD63/ CD81/ ANXA1 non-EV: Calnexin Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293T EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type TLA-100.4 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot An

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