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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	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

	EV210302	1/2	Homo sapiens	MKN74	(d)(U)C Filtration qEV	Poças J	2023	78%
Study summary Full title Syndecan-4 is a maestro of gastric cancer cell invasion and communication that underscores poor survival. All authors Poças J, Marques C, Gomes C, Otake AH, Pinto F, Ferreira M, Silva T, Faria-Ramos I, Matos R, Ribeiro AR, Senra E, Cavadas B, Batista S, Maia J, Macedo JA, Lima L, Afonso LP, Ferreira JA, Santos LL, Polónia A, Osório H, Belting M, Reis CA, Costa-Silva B, Magalhães A Journal Proc Natl Acad Sci U S A Abstract Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options (show more...)Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options. Here, we show that syndecan-4 (SDC4), a transmembrane proteoglycan, is highly expressed in intestinal subtype gastric tumors and that this signature associates with patient poor survival. Further, we mechanistically demonstrate that SDC4 is a master regulator of gastric cancer cell motility and invasion. We also find that SDC4 decorated with heparan sulfate is efficiently sorted in extracellular vesicles (EVs). Interestingly, SDC4 in EVs regulates gastric cancer cell-derived EV organ distribution, uptake, and functional effects in recipient cells. Specifically, we show that knockout disrupts the tropism of EVs for the common gastric cancer metastatic sites. Our findings set the basis for the molecular implications of SDC4 expression in gastric cancer cells and provide broader perspectives on the development of therapeutic strategies targeting the glycan-EV axis to limit tumor progression. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration qEV Protein markers EV: Alix/ CD9/ CD63/ CD81/ HSP70/ SDCBP/ SDC4 non-EV: CytochromeC/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Albumin/ Argonaute2/ Tubulin1 Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MKN74 EV-harvesting Medium Serum free medium Cell viability (%) 88 Cell count 73000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 160 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 30 Wash: time (min) 160 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Filtration steps 0.2 or 0.22 µm Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per million cells Western Blot 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% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin SDC4 KO Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration qEV Protein markers EV: Alix/ CD9/ CD81/ HSP70/ SDCBP non-EV: CytochromeC/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Albumin/ Argonaute2/ Tubulin1 Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MKN74 EV-harvesting Medium Serum free medium Cell viability (%) 92 Cell count 74000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 160 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 30 Wash: time (min) 160 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Filtration steps 0.2 or 0.22 µm Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per million cells Western Blot 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

	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

	EV220412	1/1	Bos taurus	ovary-derived granulosa cells	(d)(U)C Filtration	Menjivar, Nico G	2023	67%
Study summary Full title Granulosa cell-derived extracellular vesicles mitigate the detrimental impact of thermal stress on bovine oocytes and embryos All authors Nico G. Menjivar, Ahmed Gad, Samuel Gebremedhn, Soham Ghosh and Dawit Tesfaye Journal Front Cell Dev Biol Abstract Climate change-induced global warming results in rises in body temperatures above normal physiologic (show more...)Climate change-induced global warming results in rises in body temperatures above normal physiological levels (hyperthermia) with negative impacts on reproductive function in dairy and beef animals. Extracellular vesicles (EVs), commonly described as nano-sized, lipid-enclosed complexes, harnessed with a plethora of bioactive cargoes (RNAs, proteins, and lipids), are crucial to regulating processes like folliculogenesis and the initiation of different signaling pathways. The beneficial role of follicular fluid-derived EVs in inducing thermotolerance to oocytes during in vitro maturation (IVM) has been evidenced. Here we aimed to determine the capacity of in vitro cultured granulosa cell-derived EVs (GC-EVs) to modulate bovine oocytes’ thermotolerance to heat stress (HS) during IVM. Moreover, this study tested the hypothesis that EVs released from thermally stressed GCs (S-EVs) shuttle protective messages to provide protection against subsequent HS in bovine oocytes. For this, sub-populations of GC-EVs were generated from GCs subjected to 38.5°C (N-EVs) or 42°C (S-EVs) and supplemented to cumulus-oocyte complexes (COCs) matured in vitro at the normal physiological body temperature of the cow (38.5°C) or HS (41°C) conditions. Results indicate that S-EVs improve the survival of oocytes by reducing ROS accumulation, improving mitochondrial function, and suppressing the expression of stress-associated genes thereby reducing the severity of HS on oocytes. Moreover, our findings indicate a carryover impact from the addition of GC-EVs during oocyte maturation in the development to the blastocyst stage with enhanced viability. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: CD63/ CD81/ TSG101 non-EV: CYCS Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer Sample Species Bos taurus Sample Type Cell culture supernatant EV-producing cells ovary-derived granulosa cells EV-harvesting Medium EV-depleted medium Cell count 250000 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 Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 3.5 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ TSG101 Not detected contaminants CYCS Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR/ Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 137.75 EV concentration Yes Particle yield as numer of particles per mililiter: 1.02E+11 EM EM-type Transmission-EM Image type Close-up Report size (nm) 137.75

	EV220412	2/1	Bos taurus	ovary-derived granulosa cells	(d)(U)C Filtration	Menjivar, Nico G	2023	67%
Study summary Full title Granulosa cell-derived extracellular vesicles mitigate the detrimental impact of thermal stress on bovine oocytes and embryos All authors Nico G. Menjivar, Ahmed Gad, Samuel Gebremedhn, Soham Ghosh and Dawit Tesfaye Journal Front Cell Dev Biol Abstract Climate change-induced global warming results in rises in body temperatures above normal physiologic (show more...)Climate change-induced global warming results in rises in body temperatures above normal physiological levels (hyperthermia) with negative impacts on reproductive function in dairy and beef animals. Extracellular vesicles (EVs), commonly described as nano-sized, lipid-enclosed complexes, harnessed with a plethora of bioactive cargoes (RNAs, proteins, and lipids), are crucial to regulating processes like folliculogenesis and the initiation of different signaling pathways. The beneficial role of follicular fluid-derived EVs in inducing thermotolerance to oocytes during in vitro maturation (IVM) has been evidenced. Here we aimed to determine the capacity of in vitro cultured granulosa cell-derived EVs (GC-EVs) to modulate bovine oocytes’ thermotolerance to heat stress (HS) during IVM. Moreover, this study tested the hypothesis that EVs released from thermally stressed GCs (S-EVs) shuttle protective messages to provide protection against subsequent HS in bovine oocytes. For this, sub-populations of GC-EVs were generated from GCs subjected to 38.5°C (N-EVs) or 42°C (S-EVs) and supplemented to cumulus-oocyte complexes (COCs) matured in vitro at the normal physiological body temperature of the cow (38.5°C) or HS (41°C) conditions. Results indicate that S-EVs improve the survival of oocytes by reducing ROS accumulation, improving mitochondrial function, and suppressing the expression of stress-associated genes thereby reducing the severity of HS on oocytes. Moreover, our findings indicate a carryover impact from the addition of GC-EVs during oocyte maturation in the development to the blastocyst stage with enhanced viability. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Heat stress 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: CD63/ CD81/ TSG101 non-EV: CYCS Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer Sample Species Bos taurus Sample Type Cell culture supernatant EV-producing cells ovary-derived granulosa cells EV-harvesting Medium EV-depleted medium Cell count 250000 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 Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 3.5 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ TSG101 Not detected contaminants CYCS Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR/ Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 147.95 EV concentration Yes Particle yield as numer of particles per mililiter: 1.51E+11 EM EM-type Transmission-EM Image type Close-up Report size (nm) 147.95

	EV220409	1/2	Schistosoma mansoni	whole parasite culture	(d)(U)C DG	Kuipers ME	2023	67%
Study summary Full title Life stage-specific glycosylation of extracellular vesicles from schistosomula and adult worms drives differential interaction with C-type lectin receptors DC-SIGN and MGL. All authors Kuipers ME, Nguyen DL, van Diepen A, Mes L, Bos E, Koning RI, Nolte-'t Hoen ENM, Smits HH, Hokke CH Journal Front Mol Biosci Abstract Schistosomes can survive in mammalian hosts for many years, and this is facilitated by released para (show more...)Schistosomes can survive in mammalian hosts for many years, and this is facilitated by released parasite products that modulate the host's immune system. Many of these products are glycosylated and interact with host cells C-type lectin receptors (CLRs). We previously reported on specific fucose-containing glycans present on extracellular vesicles (EVs) released by schistosomula, the early juvenile life stage of the schistosome, and the interaction of these EVs with the C-type lectin receptor Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN or CD209). EVs are membrane vesicles with a size range between 30-1,000 nm that play a role in intercellular and interspecies communication. Here, we studied the glycosylation of EVs released by the adult schistosome worms. Mass spectrometric analysis showed that GalNAcβ1-4GlcNAc (LacDiNAc or LDN) containing N-glycans were the dominant glycan type present on adult worm EVs. Using glycan-specific antibodies, we confirmed that EVs from adult worms were predominantly associated with LDN, while schistosomula EVs displayed a highly fucosylated glycan profile. In contrast to schistosomula EV that bind to DC-SIGN, adult worm EVs are recognized by macrophage galactose-type lectin (MGL or CD301), and not by DC-SIGN, on CLR expressing cell lines. The different glycosylation profiles of adult worm- and schistosomula-derived EVs match with the characteristic glycan profiles of the corresponding life stages and support their distinct roles in schistosome life-stage specific interactions with the host. (hide) EV-METRIC 67% (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 whole parasite culture Sample origin adult worm Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: S. mansoni TSP2 non-EV: None Proteomics no EV density (g/ml) 1.09-1.18 Show all info Study aim Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Schistosoma mansoni Sample Type whole parasite culture Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 10% Highest density fraction 41.60% Total gradient volume, incl. sample (mL) 2.113 Sample volume (mL) 0.293 Orientation Bottom-up Speed (g) 166,18 Duration (min) 120 Fraction volume (mL) 0.1 Fraction processing Centrifugation Pelleting: volume per fraction 2.75 Pelleting: speed (g) 187,813 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) yes, per 100 adult worms Western Blot Antibody details provided? No Detected EV-associated proteins S. mansoni TSP2 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-250 EV concentration Yes Particle yield number per 100 adult worms EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 30-180 Extra information All data on protein concentration and NTA of the schistosomula EVs can be found in EV track ID EV190032. More information on the protocol for the cryo EM of this publication can be found in EV track ID EV220119. Furthermore, we performed glycomics, western blots targeting glycan structures, and lectin blots of the adult worm EVs. The western blots targeting glycan structures were also performed on the schistosomula EV (glycomics on these EV are linked to EV track ID EV190032).

	EV220366	1/11	Mus musculus	bone marrow-derived cells	(d)(U)C	Sako Y	2023	67%
Study summary Full title Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx. All authors Sako Y, Sato-Kaneko F, Shukla NM, Yao S, Belsuzarri MM, Chan M, Saito T, Lao FS, Kong H, Puffer M, Messer K, Pu M, Cottam HB, Carson DA, Hayashi T Journal ACS Chem Biol Abstract Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapse (show more...)Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca elevation enhances EV release, we screened 80 hit compounds and identified compound as a Ca influx inducer. enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from -treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of . The analogues that retained the ability to induce Ca influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca influx. The levels of Ca induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, , enhances the release of EVs with immunostimulatory potency via induction of Ca influx. This agent is a novel tool for EV-based immune studies and vaccine development. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD81/ CD86/ CD80/ MHCII/ CD40 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells bone marrow-derived cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell count 3.00E+07 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 28 Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 30 Wash: time (min) 180 Wash: Rotor Type SW 28 Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD81/ CD86/ CD80/ MHCII Not detected EV-associated proteins CD40 Detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 75-300 EV concentration Yes Particle yield particles per milliliter of starting sample: 5.50E+11 EM EM-type Transmission-EM Image type Wide-field

	EV220366	3/11	Mus musculus	bone marrow-derived cells	(d)(U)C	Sako Y	2023	67%
Study summary Full title Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx. All authors Sako Y, Sato-Kaneko F, Shukla NM, Yao S, Belsuzarri MM, Chan M, Saito T, Lao FS, Kong H, Puffer M, Messer K, Pu M, Cottam HB, Carson DA, Hayashi T Journal ACS Chem Biol Abstract Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapse (show more...)Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca elevation enhances EV release, we screened 80 hit compounds and identified compound as a Ca influx inducer. enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from -treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of . The analogues that retained the ability to induce Ca influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca influx. The levels of Ca induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, , enhances the release of EVs with immunostimulatory potency via induction of Ca influx. This agent is a novel tool for EV-based immune studies and vaccine development. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Ionomycin 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/ CD81/ CD86/ CD80/ MHCII/ CD40 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells bone marrow-derived cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell count 3.00E+07 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 28 Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 30 Wash: time (min) 180 Wash: Rotor Type SW 28 Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD81/ CD86/ CD80/ MHCII Not detected EV-associated proteins CD40 Detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 75-300 EV concentration Yes Particle yield particles per milliliter of starting sample: 7.50E+11 EM EM-type Transmission-EM Image type Wide-field

	EV220366	4/11	Mus musculus	bone marrow-derived cells	(d)(U)C	Sako Y	2023	67%
Study summary Full title Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx. All authors Sako Y, Sato-Kaneko F, Shukla NM, Yao S, Belsuzarri MM, Chan M, Saito T, Lao FS, Kong H, Puffer M, Messer K, Pu M, Cottam HB, Carson DA, Hayashi T Journal ACS Chem Biol Abstract Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapse (show more...)Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca elevation enhances EV release, we screened 80 hit compounds and identified compound as a Ca influx inducer. enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from -treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of . The analogues that retained the ability to induce Ca influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca influx. The levels of Ca induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, , enhances the release of EVs with immunostimulatory potency via induction of Ca influx. This agent is a novel tool for EV-based immune studies and vaccine development. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin compound 634 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/ CD81/ CD86/ CD80/ MHCII/ CD40 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells bone marrow-derived cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell count 3.00E+07 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 28 Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 30 Wash: time (min) 180 Wash: Rotor Type SW 28 Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD81/ CD86/ CD80/ MHCII Not detected EV-associated proteins CD40 Detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 75-300 EV concentration Yes Particle yield particles per milliliter of starting sample: 8.00E+11 EM EM-type Transmission-EM Image type Wide-field

	EV210141	1/5	Homo sapiens	human umbilical vein endothelial cells	IAF Ultrafiltratrion (d)(U)C	Zhao F	2023	67%
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 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Immunoaffinity capture (non-commercial) Ultrafiltratrion (Differential) (ultra)centrifugation 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 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

	EV210077	4/6	Homo sapiens	Serum	(d)(U)C	Lapitz A	2023	67%
Study summary Full title Liquid biopsy-based protein biomarkers for risk prediction, early diagnosis and prognostication of cholangiocarcinoma. All authors Lapitz A, Azkargorta M, Milkiewicz P, Olaizola P, Zhuravleva E, Grimsrud MM, Schramm C, Arbelaiz A, O'Rourke CJ, La Casta A, Milkiewicz M, Pastor T, Vesterhus M, Jimenez-Agüero R, Dill MT, Lamarca A, Valle JW, Macias RIR, Izquierdo-Sanchez L, Castaño YP, Caballero-Camino FJ, Riaño I, Krawczyk M, Ibarra C, Bustamante J, Nova-Camacho LM, Falcon-Perez JM, Elortza F, Perugorria MJ, Andersen JB, Bujanda L, Karlsen TH, Folseraas T, Rodrigues PM, Banales JM Journal J Hepatol Abstract Cholangiocarcinoma (CCA), heterogeneous biliary tumors with dismal prognosis, lacks accurate early d (show more...)Cholangiocarcinoma (CCA), heterogeneous biliary tumors with dismal prognosis, lacks accurate early diagnostic methods, especially important for individuals at high-risk (i.e., primary sclerosing cholangitis (PSC)). Here, we searched for protein biomarkers in serum extracellular vesicles (EVs). (hide) EV-METRIC 67% (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 Serum Sample origin CCA 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/ FGL1/ vWF/ PIGR/ FIBG/ FRIL/ FIBB/ CRP/ OIT3 non-EV: GRP78 Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type TLA-110 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 3 Wash: time (min) 75 Wash: Rotor Type TLA-110 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ FGL1/ vWF/ PIGR/ FIBG/ FRIL/ FIBB/ CRP/ OIT3 Not detected contaminants GRP78 Proteomics database Yes: PRIDE. Orbitrap cohort: P Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 198 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV230062	5/6	Mus musculus	Blood plasma	(d)(U)C Filtration	Choi YY	2023	63%
Study summary Full title The miR-126-5p and miR-212-3p in the extracellular vesicles activate monocytes in the early stage of radiation-induced vascular inflammation implicated in atherosclerosis. All authors Choi YY, Kim A, Lee Y, Lee YH, Park M, Shin E, Park S, Youn B, Seong KM Journal J Extracell Vesicles Abstract People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiov (show more...)People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiovascular outcomes in long-term survivors. Extracellular vesicles (EVs) are involved in radiation-induced endothelial dysfunction, but their role in the early stage of vascular inflammation after radiation exposure remains to be fully understood. Herein, we demonstrate that endothelial cell-derived EVs containing miRNAs initiate monocyte activation in radiation-induced vascular inflammation. In vitro co-culture and in vivo experimental data showed that endothelial EVs can be sensitively increased by radiation exposure in a dose-dependent manner, and stimulate monocytes releasing monocytic EVs and adhesion to endothelial cells together with an increase in the expression of genes encoding specific ligands for cell-cell interaction. Small RNA sequencing and transfection using mimics and inhibitors explained that miR-126-5p and miR-212-3p enriched in endothelial EVs initiate vascular inflammation by monocyte activation after radiation exposure. Moreover, miR-126-5p could be detected in the circulating endothelial EVs of radiation-induced atherosclerosis model mice, which was found to be tightly correlated with the atherogenic index of plasma. In summary, our study showed that miR-126-5p and miR-212-3p present in the endothelial EVs mediate the inflammatory signals to activate monocytes in radiation-induced vascular injury. A better understanding of the circulating endothelial EVs content can promote their use as diagnostic and prognostic biomarkers for atherosclerosis after radiation exposure. (hide) EV-METRIC 63% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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 Protein markers EV: CD9/ CD63/ CD81/ CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ CD45/ CD41 non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Mus musculus 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 Between 0.22 and 0.45 µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) Not reported Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Detected contaminants Calnexin/ GM130 Flow cytometry Type of Flow cytometry conventional flow cytometry Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 Antibody details provided? No Not detected EV-associated proteins CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ CD45/ CD41 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR/ RNA -sequencing Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 85 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type conventional flow cytometry Hardware adjustment Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: ~35000 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220323	2/2	Homo sapiens	Serum	exoEasy	Kim JA	2023	63%
Study summary Full title Small RNA sequencing of circulating small extracellular vesicles microRNAs in patients with amyotrophic lateral sclerosis. All authors Kim JA, Park C, Sung JJ, Seo DJ, Choi SJ, Hong YH Journal Sci Rep Abstract Dysregulation of microRNAs (miRNA) in small extracellular vesicles (sEV) such as exosomes have been (show more...)Dysregulation of microRNAs (miRNA) in small extracellular vesicles (sEV) such as exosomes have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Although circulating cell-free miRNA have been extensively investigated in ALS, sEV-derived miRNAs have not been systemically explored yet. Here, we performed small RNA sequencing analysis of serum sEV and identified 5 differentially expressed miRNA in a discovery cohort of 12 patients and 11 age- and sex-matched healthy controls (fold change > 2, p < 0.05). Two of them (up- and down-regulation of miR-23c and miR192-5p, respectively) were confirmed in a separate validation cohort (18 patients and 15 healthy controls) by droplet digital PCR. Bioinformatic analysis revealed that these two miRNAs interact with distinct sets of target genes and involve biological processes relevant to the pathomechanism of ALS. Our results suggest that circulating sEV from ALS patients have distinct miRNA profiles which may be potentially useful as a biomarker of the disease. (hide) EV-METRIC 63% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Amyotrophic lateral sclerosis 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 exoEasy Protein markers EV: CD63/ CD81 non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Serum Separation Method Commercial kit exoEasy Other Name other separation method exoEasy Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81 Not detected contaminants Calnexin/ GM130 Characterization: RNA analysis RNA analysis Type RNA-sequencing/ droplet digital PCR Database Vesiclepedia Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 138 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 138

	EV220407	1/1	Homo sapiens	adult primary astrocytes	(d)(U)C	Shanthi KB	2023	57%
Study summary Full title Human Adult Astrocyte Extracellular Vesicle Transcriptomics Study Identifies Specific RNAs Which Are Preferentially Secreted as EV Luminal Cargo. All authors Shanthi KB, Fischer D, Sharma A, Kiviniemi A, Kaakinen M, Vainio SJ, Bart G Journal Genes (Basel) Abstract Astrocytes are central nervous system (CNS)-restricted glial cells involved in synaptic function and (show more...)Astrocytes are central nervous system (CNS)-restricted glial cells involved in synaptic function and CNS blood flow regulation. Astrocyte extracellular vesicles (EVs) participate in neuronal regulation. EVs carry RNAs, either surface-bound or luminal, which can be transferred to recipient cells. We characterized the secreted EVs and RNA cargo of human astrocytes derived from an adult brain. EVs were isolated by serial centrifugation and characterized with nanoparticle tracking analysis (NTA), Exoview, and immuno-transmission electron microscopy (TEM). RNA from cells, EVs, and proteinase K/RNase-treated EVs was analyzed by miRNA-seq. Human adult astrocyte EVs ranged in sizes from 50 to 200 nm, with CD81 as the main tetraspanin marker and larger EVs positive for integrin β1. Comparison of the RNA between the cells and EVs identified RNA preferentially secreted in the EVs. In the case of miRNAs, enrichment analysis of their mRNA targets indicates that they are good candidates for mediating EV effects on recipient cells. The most abundant cellular miRNAs were also abundant in EVs, and the majority of their mRNA targets were found to be downregulated in mRNA-seq data, but the enrichment analysis lacked neuronal specificity. Proteinase K/RNase treatment of EV-enriched preparations identified RNAs secreted independently of EVs. Comparing the distribution of cellular and secreted RNA identifies the RNAs involved in intercellular communication via EVs. (hide) EV-METRIC 57% (91st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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: CD9/ CD63/ CD81/ Syntenin non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells adult primary astrocytes EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type AH-629 (36 ml) Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Other 2 Exoview Detected EV-associated proteins CD9/ CD63/ CD81/ Syntenin Characterization: RNA analysis RNA analysis Type RNA-sequencing Proteinase treatment Yes Moment of Proteinase treatment After Proteinase type Proteinase K Proteinase concentration 660 µg/ml RNAse treatment Yes RNAse type RNase A and RNase T1 RNAse concentration 0.5 mg/ml Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 107.9 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.20E+06 EM EM-type Transmission-EM/ Immuno-EM EM protein Integrin beta1 Image type Close-up, Wide-field Report size (nm) 50 to 200 nm EV concentration Non

	EV230567	1/2	Homo sapiens	Blood plasma	(d)(U)C	Li L	2023	56%
Study summary Full title Circulating extracellular vesicles from patients with traumatic brain injury induce cerebrovascular endothelial dysfunction. All authors Li L, Li F, Bai X, Jia H, Wang C, Li P, Zhang Q, Guan S, Peng R, Zhang S, Dong JF, Zhang J, Xu X Journal Pharmacol Res Abstract Endothelial dysfunction is a key proponent of pathophysiological process of traumatic brain injury ( (show more...)Endothelial dysfunction is a key proponent of pathophysiological process of traumatic brain injury (TBI). We previously demonstrated that extracellular vesicles (EVs) released from injured brains led to endothelial barrier disruption and vascular leakage. However, the molecular mechanisms of this EV-induced endothelial dysfunction (endotheliopathy) remain unclear. Here, we enriched plasma EVs from TBI patients (TEVs), and detected high mobility group box 1 (HMGB1) exposure to 50.33 ± 10.17% of TEVs and the number of HMGB1TEVs correlated with injury severity. We then investigated for the first time the impact of TEVs on endothelial function using adoptive transfer models. We found that TEVs induced dysfunction of cultured human umbilical vein endothelial cells and mediated endothelial dysfunction in both normal and TBI mice, which were propagated through the HMGB1-activated receptor for advanced glycation end products (RAGE)/Cathepsin B signaling, and the resultant NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation and canonical caspase-1/gasdermin D (GSDMD)-dependent pyroptosis. Finally, von Willebrand factor (VWF) was detected on the surface of 77.01 ± 7.51% of HMGB1TEVs. The TEV-mediated endotheliopathy was reversed by a polyclonal VWF antibody, indicating that VWF might serve a coupling factor that tethered TEVs to ECs, thus facilitating HMGB1-induced endotheliopathy. These results suggest that circulating EVs isolated from patients with TBI alone are sufficient to induce endothelial dysfunction and contribute to secondary brain injury that are dependent on immunologically active HMGB1 exposed on their surface. This finding provided new insight for the development of potential therapeutic targets and diagnostic biomarkers for TBI. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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/ TSG101/ Calnexin non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 0.1 Wash: time (min) 60 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD81/ TSG101 Not detected EV-associated proteins Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 119.2±10.09 Particle analysis: flow cytometry Flow cytometer type FACS LSR II flow cytometer Hardware adjustment - Calibration bead size 0.5/ 0.9/ 3 EV concentration Yes Particle yield particles per milliliter of starting sample: 5.32±1.82E07 and 2.4 ± 0.69E07 EM EM-type Transmission-EM Image type Close-up

	EV230567	2/2	Homo sapiens	Blood plasma	(d)(U)C	Li L	2023	56%
Study summary Full title Circulating extracellular vesicles from patients with traumatic brain injury induce cerebrovascular endothelial dysfunction. All authors Li L, Li F, Bai X, Jia H, Wang C, Li P, Zhang Q, Guan S, Peng R, Zhang S, Dong JF, Zhang J, Xu X Journal Pharmacol Res Abstract Endothelial dysfunction is a key proponent of pathophysiological process of traumatic brain injury ( (show more...)Endothelial dysfunction is a key proponent of pathophysiological process of traumatic brain injury (TBI). We previously demonstrated that extracellular vesicles (EVs) released from injured brains led to endothelial barrier disruption and vascular leakage. However, the molecular mechanisms of this EV-induced endothelial dysfunction (endotheliopathy) remain unclear. Here, we enriched plasma EVs from TBI patients (TEVs), and detected high mobility group box 1 (HMGB1) exposure to 50.33 ± 10.17% of TEVs and the number of HMGB1TEVs correlated with injury severity. We then investigated for the first time the impact of TEVs on endothelial function using adoptive transfer models. We found that TEVs induced dysfunction of cultured human umbilical vein endothelial cells and mediated endothelial dysfunction in both normal and TBI mice, which were propagated through the HMGB1-activated receptor for advanced glycation end products (RAGE)/Cathepsin B signaling, and the resultant NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation and canonical caspase-1/gasdermin D (GSDMD)-dependent pyroptosis. Finally, von Willebrand factor (VWF) was detected on the surface of 77.01 ± 7.51% of HMGB1TEVs. The TEV-mediated endotheliopathy was reversed by a polyclonal VWF antibody, indicating that VWF might serve a coupling factor that tethered TEVs to ECs, thus facilitating HMGB1-induced endotheliopathy. These results suggest that circulating EVs isolated from patients with TBI alone are sufficient to induce endothelial dysfunction and contribute to secondary brain injury that are dependent on immunologically active HMGB1 exposed on their surface. This finding provided new insight for the development of potential therapeutic targets and diagnostic biomarkers for TBI. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Traumatic brain injury 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/ TSG101/ Calnexin non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 0.1 Wash: time (min) 60 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD81/ TSG101 Not detected EV-associated proteins Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 118.4±6.98 Particle analysis: flow cytometry Flow cytometer type FACS LSR II flow cytometer Hardware adjustment nanoscale flow cytometry Calibration bead size 0.5/ 0.9/ 3 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.4±0.69E07 EM EM-type Transmission-EM Image type Close-up

	EV230053	1/2	Homo sapiens	Blood plasma	(d)(U)C	Bettio V	2023	56%
Study summary Full title Extracellular vesicles from human plasma for biomarkers discovery: Impact of anticoagulants and isolation techniques. All authors Bettio V, Mazzucco E, Antona A, Cracas S, Varalda M, Venetucci J, Bruno S, Chiabotto G, Venegoni C, Vasile A, Chiocchetti A, Quaglia M, Camussi G, Cantaluppi V, Panella M, Rolla R, Manfredi M, Capello D Journal PLoS One Abstract Extracellular vesicles (EVs) isolated from plasma are increasingly recognized as promising circulati (show more...)Extracellular vesicles (EVs) isolated from plasma are increasingly recognized as promising circulating biomarkers for disease discovery and progression, as well as for therapeutic drug delivery. The scientific community underlined the necessity of standard operative procedures for the isolation and storage of the EVs to ensure robust results. The understanding of the impact of the pre-analytical variables is still limited and some considerations about plasma anticoagulants and isolation methods are necessary. Therefore, we performed a comparison study between EVs isolated by ultracentrifugation and by affinity substrate separation from plasma EDTA and sodium citrate. The EVs were characterized by Nano Tracking Analysis, Western Blot, cytofluorimetric analysis of surface markers, and lipidomic analysis. While anticoagulants did not significantly alter any of the analyzed parameters, the isolation methods influenced EVs size, purity, surface markers expression and lipidomic profile. Compared to ultracentrifugation, affinity substrate separation yielded bigger particles highly enriched in tetraspanins (CD9, CD63, CD81), fatty acids and glycerolipids, with a predominant LDL- and vLDL-like contamination. Herein, we highlighted that the isolation method should be carefully evaluated prior to study design and the need of standardized operative procedures for EVs isolation and application to biomarkers discovery. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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/ HSP70/ MHC1 non-EV: Histones/ Albumin/ APOB48/B100/ APOA1 Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 146 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/ HSP70 Detected contaminants Albumin/ APOB48/B100/ APOA1 Not detected contaminants Histones Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81/ MHC1 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) CD9/ CD63/ CD81 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-170 EV concentration Yes Particle yield particles per milliliter of starting sample: 1000000-25000000

	EV230053	2/2	Homo sapiens	Blood plasma	(d)(U)C	Bettio V	2023	56%
Study summary Full title Extracellular vesicles from human plasma for biomarkers discovery: Impact of anticoagulants and isolation techniques. All authors Bettio V, Mazzucco E, Antona A, Cracas S, Varalda M, Venetucci J, Bruno S, Chiabotto G, Venegoni C, Vasile A, Chiocchetti A, Quaglia M, Camussi G, Cantaluppi V, Panella M, Rolla R, Manfredi M, Capello D Journal PLoS One Abstract Extracellular vesicles (EVs) isolated from plasma are increasingly recognized as promising circulati (show more...)Extracellular vesicles (EVs) isolated from plasma are increasingly recognized as promising circulating biomarkers for disease discovery and progression, as well as for therapeutic drug delivery. The scientific community underlined the necessity of standard operative procedures for the isolation and storage of the EVs to ensure robust results. The understanding of the impact of the pre-analytical variables is still limited and some considerations about plasma anticoagulants and isolation methods are necessary. Therefore, we performed a comparison study between EVs isolated by ultracentrifugation and by affinity substrate separation from plasma EDTA and sodium citrate. The EVs were characterized by Nano Tracking Analysis, Western Blot, cytofluorimetric analysis of surface markers, and lipidomic analysis. While anticoagulants did not significantly alter any of the analyzed parameters, the isolation methods influenced EVs size, purity, surface markers expression and lipidomic profile. Compared to ultracentrifugation, affinity substrate separation yielded bigger particles highly enriched in tetraspanins (CD9, CD63, CD81), fatty acids and glycerolipids, with a predominant LDL- and vLDL-like contamination. Herein, we highlighted that the isolation method should be carefully evaluated prior to study design and the need of standardized operative procedures for EVs isolation and application to biomarkers discovery. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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/ HSP70/ MHC1 non-EV: Histones/ Albumin/ APOB48/B100/ APOA1 Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 146 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/ HSP70 Detected contaminants Albumin/ APOB48/B100/ APOA1 Not detected contaminants Histones Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81/ MHC1 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) CD9/ CD63/ CD81 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 150-210 EV concentration Yes Particle yield particles per milliliter of starting sample: 12000000-68000000

	EV220406	1/1	Equus caballus	Follicular fluid	(d)(U)C Filtration	Gebremedhn S	2023	56%
Study summary Full title Dynamics of extracellular vesicle-coupled microRNAs in equine follicular fluid associated with follicle selection and ovulation. All authors Gebremedhn S, Gad A, Ishak GM, Menjivar NG, Gastal MO, Feugang JM, Prochazka R, Tesfaye D, Gastal EL Journal Mol Hum Reprod Abstract Innumerable similarities in reproductive cyclicity and hormonal alterations highlight the considerab (show more...)Innumerable similarities in reproductive cyclicity and hormonal alterations highlight the considerable utility of the mare to study aspects of follicular dynamics and reproductive function in view of the largely constricted, human research subjects. The bi-directional communication between the growing oocyte and the surrounding somatic cells embodies the hallmark of mammalian follicular development, partially mediated by extracellular vesicles (EVs) encapsulated with microRNAs (miRNAs) and present in the follicular fluid (FF). Here, we aimed to decipher the dynamics of the miRNAs in EVs from equine FF aspirated in vivo during different stages of follicular development, namely, predeviation (PreDev/ 18-20 mm), deviation (Dev/ 22-25 mm), postdeviation (PostDev/ 26-29 mm), preovulatory (PreOV/ 30-35 mm), and impending ovulation (IMP/ ∼40 mm). Approximately 176 known miRNAs were found in all groups with 144 mutually detected among all groups. Cluster analysis exhibited 15 different expression patterns during follicular development. Among these patterns, a group of 22 miRNAs (including miR-146b-5p, miR-140, and miR-143) exhibited a sharp reduction in expression from the PreDev until the PreOV stage. Another cluster of 23 miRNAs (including miR-106b, miR-199a-5p, and miR-125a-5p) exhibited a stable expression pattern at the PreDev stage until the PostDev stage, with a significant increase at the PreOV stage followed by a significant decrease at the IMP stage. In conclusion, this study provides greater insights into the stage-specific expression dynamics of FF EV-miRNAs during equine follicular development, which may propose novel approaches to improve ART and provide new biomarkers to facilitate the assessment of ovarian pathophysiological conditions. (hide) EV-METRIC 56% (86th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Follicular fluid Sample origin Control condition Focus vesicles 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: CD63/ TSG101/ Flotillin-1 non-EV: CytC Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Equus caballus Sample Type Follicular fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 120,000 Wash: volume per pellet (ml) 2 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 120,000 Filtration steps 0.2 or 0.22 ?m Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ TSG101/ Flotillin-1 Not detected contaminants CytC Characterization: RNA analysis RNA analysis Type RNA-sequencing Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield particles per milliliter of starting sample: 1.50E+08 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 91-111

	EV230005	1/4	Homo sapiens	Serum	(d)(U)C	Dobra G	2023	55%
Study summary Full title MMP-9 as Prognostic Marker for Brain Tumours: A Comparative Study on Serum-Derived Small Extracellular Vesicles. All authors Dobra G, Gyukity-Sebestyén E, Bukva M, Harmati M, Nagy V, Szabó Z, Pankotai T, Klekner Á, Buzás K Journal Cancers (Basel) Abstract Matrix metalloproteinase-9 (MMP-9) degrades the extracellular matrix, contributes to tumour cell inv (show more...)Matrix metalloproteinase-9 (MMP-9) degrades the extracellular matrix, contributes to tumour cell invasion and metastasis, and its elevated level in brain tumour tissues indicates poor prognosis. High-risk tissue biopsy can be replaced by liquid biopsy/ however, the blood-brain barrier (BBB) prevents tumour-associated components from entering the peripheral blood, making the development of blood-based biomarkers challenging. Therefore, we examined the MMP-9 content of small extracellular vesicles (sEVs)-which can cross the BBB and are stable in body fluids-to characterise tumours with different invasion capacity. From four patient groups (glioblastoma multiforme, brain metastases of lung cancer, meningioma, and lumbar disc herniation as controls), 222 serum-derived sEV samples were evaluated. After isolating and characterising sEVs, their MMP-9 content was measured by ELISA and assessed statistically (correlation, paired -test, Welch's test, ANOVA, ROC). We found that the MMP-9 content of sEVs is independent of gender and age, but is affected by surgical intervention, treatment, and recurrence. We found a relation between low MMP-9 level in sEVs (<28 ppm) and improved survival (8-month advantage) of glioblastoma patients, and MMP-9 levels showed a positive correlation with aggressiveness. These findings suggest that vesicular MMP-9 level might be a useful prognostic marker for brain tumours. (hide) EV-METRIC 55% (91st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD5L/ MMP-9 non-EV: calnexin Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type T-1270 Pelleting: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ CD5L Not detected contaminants calnexin ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins MMP-9 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 77.3 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.44E+12 EM EM-type Transmission-EM Image type Close-up

	EV220319	3/6	Homo sapiens	Serum	(d)(U)C	Schmoldt A	2023	55%
Study summary Full title Small extracellular vesicles are released ex vivo from platelets into serum and from residual blood cells into stored plasma All authors Małgorzata S. Małys, Maximilian C. Köller, Kristin Papp, Christof Aigner, Daffodil Dioso, Patrick Mucher, Helga Schachner, Michael Bonelli, Helmuth Haslacher, Andrew J. Rees, Renate Kain Journal Journal of Extracellular Biology Abstract Small extracellular vesicles (sEV) purified from blood have great potential clinically as biomarkers (show more...)Small extracellular vesicles (sEV) purified from blood have great potential clinically as biomarkers for systemic disease; however interpretation is complicated by release of sEV ex vivo after blood taking. To quantify the problem and devise ways to minimise it, we characterised sEV in paired serum, plasma and platelet poor plasma (PPP) samples from healthy donors. Immunoblotting showed twofold greater abundance of CD9 in sEV fractions from fresh serum than from fresh plasma or PPP. MACSPlex confirmed this, and showed that proteins expressed on platelet sEV, either exclusively (CD41b, CD42a and CD62P) or more widely (HLA-ABC, CD24, CD29 and CD31) were also twofold more abundant; by contrast non-platelet proteins (including CD81) were no different. Storage of plasma (but not serum) increased abundance of platelet and selected leukocyte sEV proteins to at least that of serum, and this could be recapitulated by activating cells in fresh plasma by Ca2+, an effect abrogated in PPP. This suggests that a substantial proportion of sEV in serum and stored plasma were generated ex vivo, which is not the case for fresh plasma or PPP. Thus we provide strategies to minimise ex vivo sEV generation and criteria for identifying those that were present in vivo. (hide) EV-METRIC 55% (91st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Control condition Focus vesicles small extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD9/ CD63/ CD81 non-EV: Calnexin/ Albumin/ ApoB100 Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type P28S(SRP28SA) Swinging bucket rotor Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 32 Wash: time (min) 120 Wash: Rotor Type P28S(SRP28SA) Swinging bucket rotor Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63 Not detected EV-associated proteins CD9/ CD63 Detected contaminants Albumin Not detected contaminants Calnexin ELISA Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Detected contaminants ApoB100 Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) CD9/ CD63/ CD81/ MACSPlex exosome human kit Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 161.1 EV concentration Yes Particle yield per ml of purification EM EM-type Immuno-EM EM protein CD9 Image type Close-up

	EV230062	1/6	Homo sapiens	HUVEC/THP1 (coculture)	(d)(U)C Filtration UF	Choi YY	2023	50%
Study summary Full title The miR-126-5p and miR-212-3p in the extracellular vesicles activate monocytes in the early stage of radiation-induced vascular inflammation implicated in atherosclerosis. All authors Choi YY, Kim A, Lee Y, Lee YH, Park M, Shin E, Park S, Youn B, Seong KM Journal J Extracell Vesicles Abstract People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiov (show more...)People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiovascular outcomes in long-term survivors. Extracellular vesicles (EVs) are involved in radiation-induced endothelial dysfunction, but their role in the early stage of vascular inflammation after radiation exposure remains to be fully understood. Herein, we demonstrate that endothelial cell-derived EVs containing miRNAs initiate monocyte activation in radiation-induced vascular inflammation. In vitro co-culture and in vivo experimental data showed that endothelial EVs can be sensitively increased by radiation exposure in a dose-dependent manner, and stimulate monocytes releasing monocytic EVs and adhesion to endothelial cells together with an increase in the expression of genes encoding specific ligands for cell-cell interaction. Small RNA sequencing and transfection using mimics and inhibitors explained that miR-126-5p and miR-212-3p enriched in endothelial EVs initiate vascular inflammation by monocyte activation after radiation exposure. Moreover, miR-126-5p could be detected in the circulating endothelial EVs of radiation-induced atherosclerosis model mice, which was found to be tightly correlated with the atherogenic index of plasma. In summary, our study showed that miR-126-5p and miR-212-3p present in the endothelial EVs mediate the inflammatory signals to activate monocytes in radiation-induced vascular injury. A better understanding of the circulating endothelial EVs content can promote their use as diagnostic and prognostic biomarkers for atherosclerosis after radiation exposure. (hide) EV-METRIC 50% (86th 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 Ultrafiltration Protein markers EV: CD31/ CD105/ CD144/ CD146 non-EV: None Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HUVEC/THP1 (coculture) EV-harvesting Medium Serum-containing medium Cell viability (%) 90 Cell count 4000000 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 Between 0.22 and 0.45 µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per milliliter of starting sample Flow cytometry Type of Flow cytometry conventional flow cytometer Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 Antibody details provided? No Not detected EV-associated proteins CD31/ CD105/ CD144/ CD146 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 119 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type conventional flow cytometry Hardware adjustment Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: ~2.5E06 EM EM-type Transmission-EM Image type Wide-field

	EV230062	2/6	Homo sapiens	HUVEC/THP1 (coculture)	(d)(U)C Filtration UF	Choi YY	2023	50%
Study summary Full title The miR-126-5p and miR-212-3p in the extracellular vesicles activate monocytes in the early stage of radiation-induced vascular inflammation implicated in atherosclerosis. All authors Choi YY, Kim A, Lee Y, Lee YH, Park M, Shin E, Park S, Youn B, Seong KM Journal J Extracell Vesicles Abstract People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiov (show more...)People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiovascular outcomes in long-term survivors. Extracellular vesicles (EVs) are involved in radiation-induced endothelial dysfunction, but their role in the early stage of vascular inflammation after radiation exposure remains to be fully understood. Herein, we demonstrate that endothelial cell-derived EVs containing miRNAs initiate monocyte activation in radiation-induced vascular inflammation. In vitro co-culture and in vivo experimental data showed that endothelial EVs can be sensitively increased by radiation exposure in a dose-dependent manner, and stimulate monocytes releasing monocytic EVs and adhesion to endothelial cells together with an increase in the expression of genes encoding specific ligands for cell-cell interaction. Small RNA sequencing and transfection using mimics and inhibitors explained that miR-126-5p and miR-212-3p enriched in endothelial EVs initiate vascular inflammation by monocyte activation after radiation exposure. Moreover, miR-126-5p could be detected in the circulating endothelial EVs of radiation-induced atherosclerosis model mice, which was found to be tightly correlated with the atherogenic index of plasma. In summary, our study showed that miR-126-5p and miR-212-3p present in the endothelial EVs mediate the inflammatory signals to activate monocytes in radiation-induced vascular injury. A better understanding of the circulating endothelial EVs content can promote their use as diagnostic and prognostic biomarkers for atherosclerosis after radiation exposure. (hide) EV-METRIC 50% (86th 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 Irradiation of co-culture (1 Gy) 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 Ultrafiltration Protein markers EV: CD31/ CD105/ CD144/ CD146 non-EV: None Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HUVEC/THP1 (coculture) EV-harvesting Medium Serum-containing medium Cell viability (%) 90 Cell count 4000000 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 Between 0.22 and 0.45 µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per milliliter of starting sample Flow cytometry Type of Flow cytometry conventional flow cytometry Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 Antibody details provided? No Not detected EV-associated proteins CD31/ CD105/ CD144/ CD146 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 112 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type conventional flow cytometry Hardware adjustment Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: ~5E06 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230062	3/6	Homo sapiens	Blood plasma	(d)(U)C Filtration	Choi YY	2023	50%
Study summary Full title The miR-126-5p and miR-212-3p in the extracellular vesicles activate monocytes in the early stage of radiation-induced vascular inflammation implicated in atherosclerosis. All authors Choi YY, Kim A, Lee Y, Lee YH, Park M, Shin E, Park S, Youn B, Seong KM Journal J Extracell Vesicles Abstract People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiov (show more...)People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiovascular outcomes in long-term survivors. Extracellular vesicles (EVs) are involved in radiation-induced endothelial dysfunction, but their role in the early stage of vascular inflammation after radiation exposure remains to be fully understood. Herein, we demonstrate that endothelial cell-derived EVs containing miRNAs initiate monocyte activation in radiation-induced vascular inflammation. In vitro co-culture and in vivo experimental data showed that endothelial EVs can be sensitively increased by radiation exposure in a dose-dependent manner, and stimulate monocytes releasing monocytic EVs and adhesion to endothelial cells together with an increase in the expression of genes encoding specific ligands for cell-cell interaction. Small RNA sequencing and transfection using mimics and inhibitors explained that miR-126-5p and miR-212-3p enriched in endothelial EVs initiate vascular inflammation by monocyte activation after radiation exposure. Moreover, miR-126-5p could be detected in the circulating endothelial EVs of radiation-induced atherosclerosis model mice, which was found to be tightly correlated with the atherogenic index of plasma. In summary, our study showed that miR-126-5p and miR-212-3p present in the endothelial EVs mediate the inflammatory signals to activate monocytes in radiation-induced vascular injury. A better understanding of the circulating endothelial EVs content can promote their use as diagnostic and prognostic biomarkers for atherosclerosis after radiation exposure. (hide) EV-METRIC 50% (85th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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 Protein markers EV: CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ HLA-DR/ CD45/ CD41 non-EV: None Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer/Identification of content (omics approaches) 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 Between 0.22 and 0.45 µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) Not reported Flow cytometry Type of Flow cytometry conventional flow cytometry Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 Antibody details provided? No Not detected EV-associated proteins CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ HLA-DR/ CD45/ CD41 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 82 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type conventional flow cytometry Hardware adjustment Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: ~32000 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230062	4/6	Homo sapiens	Blood plasma	(d)(U)C Filtration	Choi YY	2023	50%
Study summary Full title The miR-126-5p and miR-212-3p in the extracellular vesicles activate monocytes in the early stage of radiation-induced vascular inflammation implicated in atherosclerosis. All authors Choi YY, Kim A, Lee Y, Lee YH, Park M, Shin E, Park S, Youn B, Seong KM Journal J Extracell Vesicles Abstract People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiov (show more...)People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiovascular outcomes in long-term survivors. Extracellular vesicles (EVs) are involved in radiation-induced endothelial dysfunction, but their role in the early stage of vascular inflammation after radiation exposure remains to be fully understood. Herein, we demonstrate that endothelial cell-derived EVs containing miRNAs initiate monocyte activation in radiation-induced vascular inflammation. In vitro co-culture and in vivo experimental data showed that endothelial EVs can be sensitively increased by radiation exposure in a dose-dependent manner, and stimulate monocytes releasing monocytic EVs and adhesion to endothelial cells together with an increase in the expression of genes encoding specific ligands for cell-cell interaction. Small RNA sequencing and transfection using mimics and inhibitors explained that miR-126-5p and miR-212-3p enriched in endothelial EVs initiate vascular inflammation by monocyte activation after radiation exposure. Moreover, miR-126-5p could be detected in the circulating endothelial EVs of radiation-induced atherosclerosis model mice, which was found to be tightly correlated with the atherogenic index of plasma. In summary, our study showed that miR-126-5p and miR-212-3p present in the endothelial EVs mediate the inflammatory signals to activate monocytes in radiation-induced vascular injury. A better understanding of the circulating endothelial EVs content can promote their use as diagnostic and prognostic biomarkers for atherosclerosis after radiation exposure. (hide) EV-METRIC 50% (85th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Irradiation of blood sample (1 Gy) 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: CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ HLA-DR/ CD45/ CD41 non-EV: None Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer/Identification of content (omics approaches) 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 Between 0.22 and 0.45 µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) Not reported Flow cytometry Type of Flow cytometry conventional flow cytometry Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 Antibody details provided? No Not detected EV-associated proteins CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ HLA-DR/ CD45/ CD41 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 88 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type conventional flow cytometry Hardware adjustment Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: ~34000 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230062	6/6	Mus musculus	Blood plasma	(d)(U)C Filtration	Choi YY	2023	50%
Study summary Full title The miR-126-5p and miR-212-3p in the extracellular vesicles activate monocytes in the early stage of radiation-induced vascular inflammation implicated in atherosclerosis. All authors Choi YY, Kim A, Lee Y, Lee YH, Park M, Shin E, Park S, Youn B, Seong KM Journal J Extracell Vesicles Abstract People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiov (show more...)People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiovascular outcomes in long-term survivors. Extracellular vesicles (EVs) are involved in radiation-induced endothelial dysfunction, but their role in the early stage of vascular inflammation after radiation exposure remains to be fully understood. Herein, we demonstrate that endothelial cell-derived EVs containing miRNAs initiate monocyte activation in radiation-induced vascular inflammation. In vitro co-culture and in vivo experimental data showed that endothelial EVs can be sensitively increased by radiation exposure in a dose-dependent manner, and stimulate monocytes releasing monocytic EVs and adhesion to endothelial cells together with an increase in the expression of genes encoding specific ligands for cell-cell interaction. Small RNA sequencing and transfection using mimics and inhibitors explained that miR-126-5p and miR-212-3p enriched in endothelial EVs initiate vascular inflammation by monocyte activation after radiation exposure. Moreover, miR-126-5p could be detected in the circulating endothelial EVs of radiation-induced atherosclerosis model mice, which was found to be tightly correlated with the atherogenic index of plasma. In summary, our study showed that miR-126-5p and miR-212-3p present in the endothelial EVs mediate the inflammatory signals to activate monocytes in radiation-induced vascular injury. A better understanding of the circulating endothelial EVs content can promote their use as diagnostic and prognostic biomarkers for atherosclerosis after radiation exposure. (hide) EV-METRIC 50% (85th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Irradiation of whole body (1 Gy) 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: CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ CD45/ CD41 non-EV: None Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Mus musculus 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 Between 0.22 and 0.45 µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) Not reported Flow cytometry Type of Flow cytometry conventional flow cytometry Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 Antibody details provided? No Not detected EV-associated proteins CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ CD45/ CD41 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR/ RNA -sequencing Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 71 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type conventional flow cytometry Hardware adjustment Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: ~39000 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220409	2/2	Schistosoma mansoni	whole parasite culture	(d)(U)C DG	Kuipers ME	2023	50%
Study summary Full title Life stage-specific glycosylation of extracellular vesicles from schistosomula and adult worms drives differential interaction with C-type lectin receptors DC-SIGN and MGL. All authors Kuipers ME, Nguyen DL, van Diepen A, Mes L, Bos E, Koning RI, Nolte-'t Hoen ENM, Smits HH, Hokke CH Journal Front Mol Biosci Abstract Schistosomes can survive in mammalian hosts for many years, and this is facilitated by released para (show more...)Schistosomes can survive in mammalian hosts for many years, and this is facilitated by released parasite products that modulate the host's immune system. Many of these products are glycosylated and interact with host cells C-type lectin receptors (CLRs). We previously reported on specific fucose-containing glycans present on extracellular vesicles (EVs) released by schistosomula, the early juvenile life stage of the schistosome, and the interaction of these EVs with the C-type lectin receptor Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN or CD209). EVs are membrane vesicles with a size range between 30-1,000 nm that play a role in intercellular and interspecies communication. Here, we studied the glycosylation of EVs released by the adult schistosome worms. Mass spectrometric analysis showed that GalNAcβ1-4GlcNAc (LacDiNAc or LDN) containing N-glycans were the dominant glycan type present on adult worm EVs. Using glycan-specific antibodies, we confirmed that EVs from adult worms were predominantly associated with LDN, while schistosomula EVs displayed a highly fucosylated glycan profile. In contrast to schistosomula EV that bind to DC-SIGN, adult worm EVs are recognized by macrophage galactose-type lectin (MGL or CD301), and not by DC-SIGN, on CLR expressing cell lines. The different glycosylation profiles of adult worm- and schistosomula-derived EVs match with the characteristic glycan profiles of the corresponding life stages and support their distinct roles in schistosome life-stage specific interactions with the host. (hide) EV-METRIC 50% (21st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type whole parasite culture Sample origin schistosomula Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: S. mansoni TSP2 non-EV: None Proteomics no EV density (g/ml) 1.09-1.18 Show all info Study aim Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Schistosoma mansoni Sample Type whole parasite culture Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 10% Highest density fraction 41.60% Total gradient volume, incl. sample (mL) 2.113 Sample volume (mL) 0.293 Orientation Bottom-up Speed (g) 166,18 Duration (min) 120 Fraction volume (mL) 0.1 Fraction processing Centrifugation Pelleting: volume per fraction 2.75 Pelleting: speed (g) 187,813 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins S. mansoni TSP2 Characterization: Lipid analysis No Characterization: Particle analysis None Extra information All data on protein concentration and NTA of the schistosomula EVs can be found in EV track ID EV190032. More information on the protocol for the cryo EM of this publication can be found in EV track ID EV220119. Furthermore, we performed glycomics, western blots targeting glycan structures, and lectin blots of the adult worm EVs. The western blots targeting glycan structures were also performed on the schistosomula EV (glycomics on these EV are linked to EV track ID EV190032).

	EV210077	1/6	Homo sapiens	Serum	(d)(U)C	Lapitz A	2023	45%
Study summary Full title Liquid biopsy-based protein biomarkers for risk prediction, early diagnosis and prognostication of cholangiocarcinoma. All authors Lapitz A, Azkargorta M, Milkiewicz P, Olaizola P, Zhuravleva E, Grimsrud MM, Schramm C, Arbelaiz A, O'Rourke CJ, La Casta A, Milkiewicz M, Pastor T, Vesterhus M, Jimenez-Agüero R, Dill MT, Lamarca A, Valle JW, Macias RIR, Izquierdo-Sanchez L, Castaño YP, Caballero-Camino FJ, Riaño I, Krawczyk M, Ibarra C, Bustamante J, Nova-Camacho LM, Falcon-Perez JM, Elortza F, Perugorria MJ, Andersen JB, Bujanda L, Karlsen TH, Folseraas T, Rodrigues PM, Banales JM Journal J Hepatol Abstract Cholangiocarcinoma (CCA), heterogeneous biliary tumors with dismal prognosis, lacks accurate early d (show more...)Cholangiocarcinoma (CCA), heterogeneous biliary tumors with dismal prognosis, lacks accurate early diagnostic methods, especially important for individuals at high-risk (i.e., primary sclerosing cholangitis (PSC)). Here, we searched for protein biomarkers in serum extracellular vesicles (EVs). (hide) EV-METRIC 45% (89th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: FGL1/ vWF/ PIGR/ FIBG/ FRIL/ FIBB/ OIT3 non-EV: None Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type TLA-110 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 3 Wash: time (min) 75 Wash: Rotor Type TLA-110 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins FGL1/ vWF/ PIGR/ FIBG/ FRIL/ FIBB/ OIT3 Not detected EV-associated proteins CRP Proteomics database Yes: PRIDE. Orbitrap cohort: P Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 215 EV concentration Yes

	EV230005	2/4	Homo sapiens	Serum	(d)(U)C	Dobra G	2023	44%
Study summary Full title MMP-9 as Prognostic Marker for Brain Tumours: A Comparative Study on Serum-Derived Small Extracellular Vesicles. All authors Dobra G, Gyukity-Sebestyén E, Bukva M, Harmati M, Nagy V, Szabó Z, Pankotai T, Klekner Á, Buzás K Journal Cancers (Basel) Abstract Matrix metalloproteinase-9 (MMP-9) degrades the extracellular matrix, contributes to tumour cell inv (show more...)Matrix metalloproteinase-9 (MMP-9) degrades the extracellular matrix, contributes to tumour cell invasion and metastasis, and its elevated level in brain tumour tissues indicates poor prognosis. High-risk tissue biopsy can be replaced by liquid biopsy/ however, the blood-brain barrier (BBB) prevents tumour-associated components from entering the peripheral blood, making the development of blood-based biomarkers challenging. Therefore, we examined the MMP-9 content of small extracellular vesicles (sEVs)-which can cross the BBB and are stable in body fluids-to characterise tumours with different invasion capacity. From four patient groups (glioblastoma multiforme, brain metastases of lung cancer, meningioma, and lumbar disc herniation as controls), 222 serum-derived sEV samples were evaluated. After isolating and characterising sEVs, their MMP-9 content was measured by ELISA and assessed statistically (correlation, paired -test, Welch's test, ANOVA, ROC). We found that the MMP-9 content of sEVs is independent of gender and age, but is affected by surgical intervention, treatment, and recurrence. We found a relation between low MMP-9 level in sEVs (<28 ppm) and improved survival (8-month advantage) of glioblastoma patients, and MMP-9 levels showed a positive correlation with aggressiveness. These findings suggest that vesicular MMP-9 level might be a useful prognostic marker for brain tumours. (hide) EV-METRIC 44% (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 Serum Sample origin Glioblastoma 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/ CD5L/ MMP-9 non-EV: calnexin Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type T-1270 Pelleting: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ CD5L Not detected contaminants calnexin ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins MMP-9 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 80.6 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.46E+12

	EV230005	3/4	Homo sapiens	Serum	(d)(U)C	Dobra G	2023	44%
Study summary Full title MMP-9 as Prognostic Marker for Brain Tumours: A Comparative Study on Serum-Derived Small Extracellular Vesicles. All authors Dobra G, Gyukity-Sebestyén E, Bukva M, Harmati M, Nagy V, Szabó Z, Pankotai T, Klekner Á, Buzás K Journal Cancers (Basel) Abstract Matrix metalloproteinase-9 (MMP-9) degrades the extracellular matrix, contributes to tumour cell inv (show more...)Matrix metalloproteinase-9 (MMP-9) degrades the extracellular matrix, contributes to tumour cell invasion and metastasis, and its elevated level in brain tumour tissues indicates poor prognosis. High-risk tissue biopsy can be replaced by liquid biopsy/ however, the blood-brain barrier (BBB) prevents tumour-associated components from entering the peripheral blood, making the development of blood-based biomarkers challenging. Therefore, we examined the MMP-9 content of small extracellular vesicles (sEVs)-which can cross the BBB and are stable in body fluids-to characterise tumours with different invasion capacity. From four patient groups (glioblastoma multiforme, brain metastases of lung cancer, meningioma, and lumbar disc herniation as controls), 222 serum-derived sEV samples were evaluated. After isolating and characterising sEVs, their MMP-9 content was measured by ELISA and assessed statistically (correlation, paired -test, Welch's test, ANOVA, ROC). We found that the MMP-9 content of sEVs is independent of gender and age, but is affected by surgical intervention, treatment, and recurrence. We found a relation between low MMP-9 level in sEVs (<28 ppm) and improved survival (8-month advantage) of glioblastoma patients, and MMP-9 levels showed a positive correlation with aggressiveness. These findings suggest that vesicular MMP-9 level might be a useful prognostic marker for brain tumours. (hide) EV-METRIC 44% (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 Serum Sample origin Brain metastasis originated from non-small cell lung 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 Protein markers EV: Alix/ CD5L/ MMP-9 non-EV: calnexin Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type T-1270 Pelleting: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ CD5L Not detected contaminants calnexin ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins MMP-9 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 84.8 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.59E+12

	EV230005	4/4	Homo sapiens	Serum	(d)(U)C	Dobra G	2023	44%
Study summary Full title MMP-9 as Prognostic Marker for Brain Tumours: A Comparative Study on Serum-Derived Small Extracellular Vesicles. All authors Dobra G, Gyukity-Sebestyén E, Bukva M, Harmati M, Nagy V, Szabó Z, Pankotai T, Klekner Á, Buzás K Journal Cancers (Basel) Abstract Matrix metalloproteinase-9 (MMP-9) degrades the extracellular matrix, contributes to tumour cell inv (show more...)Matrix metalloproteinase-9 (MMP-9) degrades the extracellular matrix, contributes to tumour cell invasion and metastasis, and its elevated level in brain tumour tissues indicates poor prognosis. High-risk tissue biopsy can be replaced by liquid biopsy/ however, the blood-brain barrier (BBB) prevents tumour-associated components from entering the peripheral blood, making the development of blood-based biomarkers challenging. Therefore, we examined the MMP-9 content of small extracellular vesicles (sEVs)-which can cross the BBB and are stable in body fluids-to characterise tumours with different invasion capacity. From four patient groups (glioblastoma multiforme, brain metastases of lung cancer, meningioma, and lumbar disc herniation as controls), 222 serum-derived sEV samples were evaluated. After isolating and characterising sEVs, their MMP-9 content was measured by ELISA and assessed statistically (correlation, paired -test, Welch's test, ANOVA, ROC). We found that the MMP-9 content of sEVs is independent of gender and age, but is affected by surgical intervention, treatment, and recurrence. We found a relation between low MMP-9 level in sEVs (<28 ppm) and improved survival (8-month advantage) of glioblastoma patients, and MMP-9 levels showed a positive correlation with aggressiveness. These findings suggest that vesicular MMP-9 level might be a useful prognostic marker for brain tumours. (hide) EV-METRIC 44% (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 Serum Sample origin Meningioma 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/ CD5L/ MMP-9 non-EV: calnexin Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type T-1270 Pelleting: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ CD5L Not detected contaminants calnexin ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins MMP-9 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 82.7 EV concentration Yes Particle yield particles per milliliter of starting sample: 3.61E+12

	EV220418	1/8	Mus musculus	Heart tissue	(d)(U)C	Schoger E	2023	44%
Study summary Full title Single-cell transcriptomics reveal extracellular vesicles secretion with a cardiomyocyte proteostasis signature during pathological remodeling. All authors Schoger E, Bleckwedel F, Germena G, Rocha C, Tucholla P, Sobitov I, Möbius W, Sitte M, Lenz C, Samak M, Hinkel R, Varga ZV, Giricz Z, Salinas G, Gross JC, Zelarayán LC Journal Commun Biol Abstract Aberrant Wnt activation has been reported in failing cardiomyocytes. Here we present single cell tr (show more...)Aberrant Wnt activation has been reported in failing cardiomyocytes. Here we present single cell transcriptome profiling of hearts with inducible cardiomyocyte-specific Wnt activation (β-cat) as well as with compensatory and failing hypertrophic remodeling. We show that functional enrichment analysis points to an involvement of extracellular vesicles (EVs) related processes in hearts of β-cat mice. A proteomic analysis of in vivo cardiac derived EVs from β-cat hearts has identified differentially enriched proteins involving 20 S proteasome constitutes, protein quality control (PQC), chaperones and associated cardiac proteins including α-Crystallin B (CRYAB) and sarcomeric components. The hypertrophic model confirms that cardiomyocytes reacted with an acute early transcriptional upregulation of exosome biogenesis processes and chaperones transcripts including CRYAB, which is ameliorated in advanced remodeling. Finally, human induced pluripotent stem cells (iPSC)-derived cardiomyocytes subjected to pharmacological Wnt activation recapitulated the increased expression of exosomal markers, CRYAB accumulation and increased PQC signaling. These findings reveal that secretion of EVs with a proteostasis signature contributes to early patho-physiological adaptation of cardiomyocytes, which may serve as a read-out of disease progression and can be used for monitoring cellular remodeling in vivo with a possible diagnostic and prognostic role in the future. (hide) EV-METRIC 44% (35th 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,

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