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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220194	3/6	Mus musculus	Liver tissue	(d)(U)C Filtration	Matejovič A	2021	67%
Study summary Full title Comparison of separation methods for tissue-derived extracellular vesicles in the liver, heart, and skeletal muscle. All authors Matejovič A, Wakao S, Kitada M, Kushida Y, Dezawa M Journal FEBS Open Bio Abstract Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messen (show more...)Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messengers, have high potential as biomarkers. EVs are usually collected from in vitro sources, such as cell culture media or biofluids, and not from tissues. Techniques enabling direct collection of EVs from tissues will extend the applications of EVs. We compared methods for separating EVs from solid liver, heart, and skeletal muscle. Compared with a precipitation method, an ultracentrifugation-based method for collection of EVs from solid tissues yielded a higher proportion of EVs positive for EV-related markers, with minimum levels of intracellular organelle-related markers. Some tissue-specific modifications, such as a sucrose cushion step, may improve the yield and purity of the collected EVs. (hide) EV-METRIC 67% (83rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Liver tissue Sample origin Cut liver - CCL4-induced hepatotoxicity Focus vesicles 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 Filtration Protein markers EV: Alix/ CD63/ HSP70/ TSG101 non-EV: calnexin/ RPL5 Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Liver tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 210053 Filtration steps 0.2 or 0.22 µm Other Name other separation method Filtration 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 Alix/ CD63/ HSP70/ TSG101 Not detected contaminants calnexin/ RPL5 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 117 ± 40.6 nm and 287 ± 65.9 nm EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Extra information Tissue processing: enzymatic digestion (2 mg/mL collagenase D and 40 U/mL DNase I) and 30 min incubation at 37 Celsius degrees/ tissue cutting (1x1 mm pieces)

	EV220194	4/6	Mus musculus	Skeletal muscle tissue	(d)(U)C Filtration	Matejovič A	2021	67%
Study summary Full title Comparison of separation methods for tissue-derived extracellular vesicles in the liver, heart, and skeletal muscle. All authors Matejovič A, Wakao S, Kitada M, Kushida Y, Dezawa M Journal FEBS Open Bio Abstract Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messen (show more...)Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messengers, have high potential as biomarkers. EVs are usually collected from in vitro sources, such as cell culture media or biofluids, and not from tissues. Techniques enabling direct collection of EVs from tissues will extend the applications of EVs. We compared methods for separating EVs from solid liver, heart, and skeletal muscle. Compared with a precipitation method, an ultracentrifugation-based method for collection of EVs from solid tissues yielded a higher proportion of EVs positive for EV-related markers, with minimum levels of intracellular organelle-related markers. Some tissue-specific modifications, such as a sucrose cushion step, may improve the yield and purity of the collected EVs. (hide) EV-METRIC 67% (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 Skeletal muscle tissue Sample origin Cut skeletal muscle - cardiotoxin-induced injury Focus vesicles 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 Filtration Protein markers EV: Alix/ CD63/ HSP70/ TSG101 non-EV: RPL5/ calnexin Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Skeletal muscle tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 210053 Filtration steps 0.2 or 0.22 µm Other Name other separation method Filtration 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 Alix/ CD63/ HSP70/ TSG101 Detected contaminants calnexin Not detected contaminants RPL5 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) a proportion of large EVs (260 ± 80.8 nm) among the small EVs (88 ± 35.5 nm) EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Extra information Tissue processing: enzymatic digestion (2 mg/mL collagenase D and 40 U/mL DNase I) and 30 min incubation at 37 Celsius degrees/ tissue cutting (1x1 mm pieces)

	EV220194	5/6	Mus musculus	Heart tissue	(d)(U)C Filtration	Matejovič A	2021	67%
Study summary Full title Comparison of separation methods for tissue-derived extracellular vesicles in the liver, heart, and skeletal muscle. All authors Matejovič A, Wakao S, Kitada M, Kushida Y, Dezawa M Journal FEBS Open Bio Abstract Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messen (show more...)Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messengers, have high potential as biomarkers. EVs are usually collected from in vitro sources, such as cell culture media or biofluids, and not from tissues. Techniques enabling direct collection of EVs from tissues will extend the applications of EVs. We compared methods for separating EVs from solid liver, heart, and skeletal muscle. Compared with a precipitation method, an ultracentrifugation-based method for collection of EVs from solid tissues yielded a higher proportion of EVs positive for EV-related markers, with minimum levels of intracellular organelle-related markers. Some tissue-specific modifications, such as a sucrose cushion step, may improve the yield and purity of the collected EVs. (hide) EV-METRIC 67% (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 Heart tissue Sample origin Cut heart - doxorubicin-induced cardiomyopathy Focus vesicles 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 Filtration Protein markers EV: Alix/ HSP70/ TSG101/ CD63 non-EV: calnexin Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Heart tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 210053 Filtration steps 0.2 or 0.22 µm 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 Alix/ HSP70/ TSG101 Not detected EV-associated proteins CD63 Detected contaminants calnexin Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 108 ± 33.5 nm and 398 ± 167.5 nm EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Extra information Tissue processing: enzymatic digestion (2 mg/mL collagenase D and 40 U/mL DNase I) and 30 min incubation at 37 Celsius degrees/ tissue cutting (1x1 mm pieces)

	EV220194	6/6	Mus musculus	Heart tissue	(d)(U)C DC Filtration	Matejovič A	2021	67%
Study summary Full title Comparison of separation methods for tissue-derived extracellular vesicles in the liver, heart, and skeletal muscle. All authors Matejovič A, Wakao S, Kitada M, Kushida Y, Dezawa M Journal FEBS Open Bio Abstract Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messen (show more...)Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messengers, have high potential as biomarkers. EVs are usually collected from in vitro sources, such as cell culture media or biofluids, and not from tissues. Techniques enabling direct collection of EVs from tissues will extend the applications of EVs. We compared methods for separating EVs from solid liver, heart, and skeletal muscle. Compared with a precipitation method, an ultracentrifugation-based method for collection of EVs from solid tissues yielded a higher proportion of EVs positive for EV-related markers, with minimum levels of intracellular organelle-related markers. Some tissue-specific modifications, such as a sucrose cushion step, may improve the yield and purity of the collected EVs. (hide) EV-METRIC 67% (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 Heart tissue Sample origin Cut heart - doxorubicin-induced cardiomyopathy Focus vesicles 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 Density cushion Filtration Protein markers EV: Alix/ CD63/ HSP70/ TSG101 non-EV: calnexin Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Heart tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 210053 Wash: volume per pellet (ml) 11 mL (PBS) Wash: time (min) 60 Wash: Rotor Type SW 41 Ti Wash: speed (g) 210053 Filtration steps 0.2 or 0.22 µm Density cushion Density medium Sucrose Sample volume 11 Cushion volume 1 Density of the cushion 30% Centrifugation time 60 Centrifugation speed 210053 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 Alix/ CD63/ HSP70/ TSG101 Detected contaminants calnexin Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 125 ± 39.8 nm and 398 ± 167.5 nm EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Extra information Tissue processing: enzymatic digestion (2 mg/mL collagenase D and 40 U/mL DNase I) and 30 min incubation at 37 Celsius degrees/ tissue cutting (1x1 mm pieces)

	EV220194	1/6	Mus musculus	Liver tissue	(d)(U)C Filtration ExoQuick	Matejovič A	2021	63%
Study summary Full title Comparison of separation methods for tissue-derived extracellular vesicles in the liver, heart, and skeletal muscle. All authors Matejovič A, Wakao S, Kitada M, Kushida Y, Dezawa M Journal FEBS Open Bio Abstract Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messen (show more...)Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messengers, have high potential as biomarkers. EVs are usually collected from in vitro sources, such as cell culture media or biofluids, and not from tissues. Techniques enabling direct collection of EVs from tissues will extend the applications of EVs. We compared methods for separating EVs from solid liver, heart, and skeletal muscle. Compared with a precipitation method, an ultracentrifugation-based method for collection of EVs from solid tissues yielded a higher proportion of EVs positive for EV-related markers, with minimum levels of intracellular organelle-related markers. Some tissue-specific modifications, such as a sucrose cushion step, may improve the yield and purity of the collected EVs. (hide) EV-METRIC 63% (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 Liver tissue Sample origin Intact liver - CCL4-induced hepatotoxicity Focus vesicles 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 Filtration ExoQuick Protein markers EV: Alix/ CD63/ HSP70/ TSG101 non-EV: Calnexin/ RPL5 Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Liver tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.2 or 0.22 µm Commercial kit ExoQuick Other Name other separation method ExoQuick 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 Alix/ CD63/ HSP70/ TSG101 Detected contaminants Calnexin Not detected contaminants Calnexin/ RPL5 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 346 ± 152.9 nm EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Extra information Tissue processing: ex vivo culture of intact tissue (48 h)

	EV220194	2/6	Mus musculus	Liver tissue	(d)(U)C Filtration ExoQuick	Matejovič A	2021	56%
Study summary Full title Comparison of separation methods for tissue-derived extracellular vesicles in the liver, heart, and skeletal muscle. All authors Matejovič A, Wakao S, Kitada M, Kushida Y, Dezawa M Journal FEBS Open Bio Abstract Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messen (show more...)Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messengers, have high potential as biomarkers. EVs are usually collected from in vitro sources, such as cell culture media or biofluids, and not from tissues. Techniques enabling direct collection of EVs from tissues will extend the applications of EVs. We compared methods for separating EVs from solid liver, heart, and skeletal muscle. Compared with a precipitation method, an ultracentrifugation-based method for collection of EVs from solid tissues yielded a higher proportion of EVs positive for EV-related markers, with minimum levels of intracellular organelle-related markers. Some tissue-specific modifications, such as a sucrose cushion step, may improve the yield and purity of the collected EVs. (hide) EV-METRIC 56% (16th 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 Liver tissue Sample origin Cut liver - CCL4-induced hepatotoxicity Focus vesicles 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 Filtration ExoQuick Protein markers EV: Alix/ CD63/ HSP70/ TSG101 non-EV: RPL5/ calnexin Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Liver tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.2 or 0.22 µm Commercial kit ExoQuick Other Name other separation method ExoQuick 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 Alix/ CD63/ HSP70/ TSG101 Detected contaminants calnexin Not detected contaminants RPL5 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 540 ± 232.7 nm EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Extra information Tissue processing: ex vivo culture of minced tissue (48 h)/ tissue cutting (1x1 mm pieces)

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EV-TRACK ID	EV220194
species	Mus musculus
sample type	Liver tissue	Skeletal muscle tissue	Heart tissue	Heart tissue	Liver tissue	Liver tissue
condition	Cut liver - CCL4-induced hepatotoxicity	Cut skeletal muscle - cardiotoxin-induced injury	Cut heart - doxorubicin-induced cardiomyopathy	Cut heart - doxorubicin-induced cardiomyopathy	Intact liver - CCL4-induced hepatotoxicity	Cut liver - CCL4-induced hepatotoxicity
separation protocol	dUC/ Filtration	dUC/ Filtration	dUC/ Filtration	dUC/ DC/ Filtration	dUC/ Filtration/ ExoQuick	dUC/ Filtration/ ExoQuick
Exp. nr.	3	4	5	6	1	2
EV-METRIC %	67	67	67	67	63	56