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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210076	1/6	Rattus norvegicus	Urine	(d)(U)C Filtration	Hinzman CP	2022	67%
Study summary Full title An optimized method for the isolation of urinary extracellular vesicles for molecular phenotyping: detection of biomarkers for radiation exposure. All authors Hinzman CP, Jayatilake M, Bansal S, Fish BL, Li Y, Zhang Y, Bansal S, Girgis M, Iliuk A, Xu X, Fernandez JA, Griffin JH, Ballew EA, Unger K, Boerma M, Medhora M, Cheema AK Journal J Transl Med Abstract Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications ac (show more...)Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications across clinical research, including monitoring radiation exposure. A key limitation to their implementation is minimal standardization in EV isolation and analytical methods. Further, most urinary EV isolation protocols necessitate large volumes of sample. This study aimed to compare and optimize isolation and analytical methods for EVs from small volumes of urine. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin 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: TSG101/ ANXA5/ CD81/ Alix/ Flotillin1/ EpCAM/ CD63 non-EV: GM130 Proteomics no Show all info Study aim New methodological development/Biomarker/Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 120.000 Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Resin type 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 Flotillin1/ CD63/ EpCAM/ ANXA5/ TSG101/ Alix/ CD81 Detected contaminants GM130 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 135 EV concentration Yes EM EM-type Cryo-EM Image type Close-up

	EV210076	4/6	Rattus norvegicus	Urine	(d)(U)C Filtration	Hinzman CP	2022	67%
Study summary Full title An optimized method for the isolation of urinary extracellular vesicles for molecular phenotyping: detection of biomarkers for radiation exposure. All authors Hinzman CP, Jayatilake M, Bansal S, Fish BL, Li Y, Zhang Y, Bansal S, Girgis M, Iliuk A, Xu X, Fernandez JA, Griffin JH, Ballew EA, Unger K, Boerma M, Medhora M, Cheema AK Journal J Transl Med Abstract Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications ac (show more...)Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications across clinical research, including monitoring radiation exposure. A key limitation to their implementation is minimal standardization in EV isolation and analytical methods. Further, most urinary EV isolation protocols necessitate large volumes of sample. This study aimed to compare and optimize isolation and analytical methods for EVs from small volumes of urine. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin 13 Gy ionizing radiation 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: TSG101/ CD63/ CD81/ ANXA5/ Alix/ Flotillin1/ EpCam non-EV: GM130 Proteomics no Show all info Study aim New methodological development/Biomarker/Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 120.000 Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Resin type 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 Flotillin1/ Alix/ EpCam/ ANXA5/ CD63/ TSG101/ CD81 Detected contaminants GM130 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 134.9 EV concentration Yes EM EM-type Cryo-EM Image type Close-up

	EV210076	2/6	Rattus norvegicus	Urine	UF qEV SEC (non-commercial)	Hinzman CP	2022	63%
Study summary Full title An optimized method for the isolation of urinary extracellular vesicles for molecular phenotyping: detection of biomarkers for radiation exposure. All authors Hinzman CP, Jayatilake M, Bansal S, Fish BL, Li Y, Zhang Y, Bansal S, Girgis M, Iliuk A, Xu X, Fernandez JA, Griffin JH, Ballew EA, Unger K, Boerma M, Medhora M, Cheema AK Journal J Transl Med Abstract Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications ac (show more...)Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications across clinical research, including monitoring radiation exposure. A key limitation to their implementation is minimal standardization in EV isolation and analytical methods. Further, most urinary EV isolation protocols necessitate large volumes of sample. This study aimed to compare and optimize isolation and analytical methods for EVs from small volumes of urine. (hide) EV-METRIC 63% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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 Ultrafiltration Commercial method Size-exclusion chromatography (non-commercial) Protein markers EV: TSG101/ ANXA5/ CD81/ Alix/ Flotillin1/ EpCAM/ CD63 non-EV: GM130 Proteomics no Show all info Study aim New methodological development/Biomarker/Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Urine Separation Method Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Commercial kit qEV Size-exclusion chromatography Total column volume (mL) 3.5 Sample volume/column (mL) 0.15 Resin type Not Specified 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 Flotillin1/ CD63/ EpCAM/ ANXA5/ TSG101/ Alix/ CD81 Detected contaminants GM130 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 156.5 EV concentration Yes EM EM-type Cryo-EM Image type Close-up

	EV210076	3/6	Rattus norvegicus	Urine	Other/ EVTrap	Hinzman CP	2022	63%
Study summary Full title An optimized method for the isolation of urinary extracellular vesicles for molecular phenotyping: detection of biomarkers for radiation exposure. All authors Hinzman CP, Jayatilake M, Bansal S, Fish BL, Li Y, Zhang Y, Bansal S, Girgis M, Iliuk A, Xu X, Fernandez JA, Griffin JH, Ballew EA, Unger K, Boerma M, Medhora M, Cheema AK Journal J Transl Med Abstract Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications ac (show more...)Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications across clinical research, including monitoring radiation exposure. A key limitation to their implementation is minimal standardization in EV isolation and analytical methods. Further, most urinary EV isolation protocols necessitate large volumes of sample. This study aimed to compare and optimize isolation and analytical methods for EVs from small volumes of urine. (hide) EV-METRIC 63% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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 Commercial method Protein markers EV: TSG101/ CD63/ CD81/ Alix/ Flotillin1/ EpCam/ ANXA5 non-EV: GM130 Proteomics no Show all info Study aim New methodological development/Biomarker/Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Urine Separation Method Commercial kit Other/ EVTrap Size-exclusion chromatography Resin type 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 Flotillin1/ Alix/ CD63/ TSG101/ EpCam/ ANXA5/ CD81 Detected contaminants GM130 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 115.9 EV concentration Yes EM EM-type Cryo-EM Image type Close-up

	EV210076	5/6	Rattus norvegicus	Urine	UF qEV SEC (non-commercial)	Hinzman CP	2022	63%
Study summary Full title An optimized method for the isolation of urinary extracellular vesicles for molecular phenotyping: detection of biomarkers for radiation exposure. All authors Hinzman CP, Jayatilake M, Bansal S, Fish BL, Li Y, Zhang Y, Bansal S, Girgis M, Iliuk A, Xu X, Fernandez JA, Griffin JH, Ballew EA, Unger K, Boerma M, Medhora M, Cheema AK Journal J Transl Med Abstract Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications ac (show more...)Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications across clinical research, including monitoring radiation exposure. A key limitation to their implementation is minimal standardization in EV isolation and analytical methods. Further, most urinary EV isolation protocols necessitate large volumes of sample. This study aimed to compare and optimize isolation and analytical methods for EVs from small volumes of urine. (hide) EV-METRIC 63% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin 13 Gy ionizing radiation 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 Ultrafiltration Commercial method Size-exclusion chromatography (non-commercial) Protein markers EV: TSG101/ ANXA5/ CD63/ Alix/ CD81/ Flotillin1/ EpCam non-EV: GM130 Proteomics no Show all info Study aim New methodological development/Biomarker/Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Urine Separation Method Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Commercial kit qEV Size-exclusion chromatography Total column volume (mL) 3.5 Sample volume/column (mL) 0.15 Resin type Not Specified 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 Flotillin1/ EpCam/ ANXA5/ CD63/ TSG101/ Alix/ CD81 Detected contaminants GM130 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 144.4 EV concentration Yes EM EM-type Cryo-EM Image type Close-up

	EV210076	6/6	Rattus norvegicus	Urine	Other/ EVTrap	Hinzman CP	2022	63%
Study summary Full title An optimized method for the isolation of urinary extracellular vesicles for molecular phenotyping: detection of biomarkers for radiation exposure. All authors Hinzman CP, Jayatilake M, Bansal S, Fish BL, Li Y, Zhang Y, Bansal S, Girgis M, Iliuk A, Xu X, Fernandez JA, Griffin JH, Ballew EA, Unger K, Boerma M, Medhora M, Cheema AK Journal J Transl Med Abstract Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications ac (show more...)Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications across clinical research, including monitoring radiation exposure. A key limitation to their implementation is minimal standardization in EV isolation and analytical methods. Further, most urinary EV isolation protocols necessitate large volumes of sample. This study aimed to compare and optimize isolation and analytical methods for EVs from small volumes of urine. (hide) EV-METRIC 63% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin 13 Gy ionizing radiation 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 Commercial method Protein markers EV: TSG101/ CD63/ CD81/ ANXA5/ Alix/ Flotillin1/ EpCam non-EV: GM130 Proteomics no Show all info Study aim New methodological development/Biomarker/Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Urine Separation Method Commercial kit Other/ EVTrap 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 Flotillin1/ Alix/ EpCam/ ANXA5/ TSG101/ CD63/ CD81 Detected contaminants GM130 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 125.2 EV concentration Yes EM EM-type Cryo-EM Image type Close-up

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EV-TRACK ID	EV210076
species	Rattus norvegicus
sample type	Urine
condition	Control condition	13 Gy ionizing radiation	Control condition	Control condition	13 Gy ionizing radiation	13 Gy ionizing radiation
separation protocol	dUC/ Filtration	dUC/ Filtration	Ultrafiltration/ qEV/ Size-exclusion chromatography (non-commercial)	Other/ EVTrap	Ultrafiltration/ qEV/ Size-exclusion chromatography (non-commercial)	Other/ EVTrap
Exp. nr.	1	4	2	3	5	6
EV-METRIC %	67	67	63	63	63	63