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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200092	1/5	Homo sapiens	Urine	(d)(U)C Filtration	Dong, Liang	2021	56%
Study summary Full title Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium All authors Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue Journal J Extracell Vesicles Abstract One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (hide) EV-METRIC 56% (88th 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 (d)(U)C Filtration Protein markers EV: CD81/ Flotillin1/ CD63/ CD9 non-EV: Tamm-Horsfall protein/ Calreticulin Proteomics no 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 Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 28 Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 120000 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins Flotillin1/ CD63/ CD81 Detected contaminants Tamm-Horsfall protein Not detected contaminants Calreticulin Flow cytometry Type of Flow cytometry NanoFCM Hardware adaptation to ~100nm EV's Please refer to the following publication: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nan Calibration bead size 0.2 Detected EV-associated proteins CD63/ CD9 Not detected EV-associated proteins CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 83.5 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 3.48E+08 Particle analysis: flow cytometry Flow cytometer type NanoFCM Hardware adjustment Please refer to the following publication: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006. Calibration bead size 0.2 EV concentration Yes

	EV200092	2/5	Homo sapiens	Urine	(d)(U)C Filtration Total Exosome Isolation	Dong, Liang	2021	50%
Study summary Full title Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium All authors Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue Journal J Extracell Vesicles Abstract One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (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 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 (d)(U)C Filtration Commercial method Protein markers EV: CD81/ Flotillin1/ None/ CD63/ CD9 non-EV: Tamm-Horsfall protein/ Calreticulin Proteomics no 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 Between 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.45µm > x > 0.22µm, Commercial kit Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins None Not detected EV-associated proteins CD81/ Flotillin1/ CD63 Not detected contaminants Calreticulin/ Tamm-Horsfall protein Flow cytometry Type of Flow cytometry NanoFCM Hardware adaptation to ~100nm EV's Please refer to the following publication: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nan Calibration bead size 0.2 Detected EV-associated proteins CD63/ CD9 Not detected EV-associated proteins CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 107.5 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 6.85E+08 Particle analysis: flow cytometry Flow cytometer type NanoFCM Hardware adjustment Please refer to the following publication: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006. Calibration bead size 0.2 Report type Modus Reported size (nm) 59.75 EV concentration Yes

	EV200092	3/5	Homo sapiens	Urine	(d)(U)C Filtration Exodisc	Dong, Liang	2021	50%
Study summary Full title Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium All authors Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue Journal J Extracell Vesicles Abstract One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (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 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 (d)(U)C Filtration Exodisc Protein markers EV: CD81/ Flotillin1/ CD63/ CD9 non-EV: Tamm-Horsfall protein/ Calreticulin Proteomics no 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 Between 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.45µm > x > 0.22µm, Other Name other separation method Exodisc Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins Flotillin1/ CD63/ CD81 Not detected contaminants Calreticulin/ Tamm-Horsfall protein Flow cytometry Type of Flow cytometry NanoFCM Hardware adaptation to ~100nm EV's Please refer to the following publication: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nan Calibration bead size 0.2 Detected EV-associated proteins CD63/ CD9 Not detected EV-associated proteins CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 110.5 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 3.64E+09 Particle analysis: flow cytometry Flow cytometer type NanoFCM Hardware adjustment Please refer to the following publication: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006. Calibration bead size 0.2 Report type Modus Reported size (nm) 59.75 EV concentration Yes

	EV200092	4/5	Homo sapiens	Urine	(d)(U)C Filtration SEC and UF	Dong, Liang	2021	50%
Study summary Full title Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium All authors Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue Journal J Extracell Vesicles Abstract One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (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 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 (d)(U)C Filtration SEC and UF Protein markers EV: CD81/ Flotillin1/ CD63/ CD9 non-EV: Tamm-Horsfall protein/ Calreticulin Proteomics no 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 Between 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.45µm > x > 0.22µm, Other Name other separation method SEC and UF Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins Flotillin1/ CD63/ CD81 Not detected contaminants Calreticulin/ Tamm-Horsfall protein Flow cytometry Type of Flow cytometry NanoFCM Hardware adaptation to ~100nm EV's Please refer to the following publication: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nan Calibration bead size 0.2 Detected EV-associated proteins CD63/ CD9 Not detected EV-associated proteins CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 110.5 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2.77E+09 Particle analysis: flow cytometry Flow cytometer type NanoFCM Hardware adjustment Please refer to the following publication: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006. Calibration bead size 0.2 Report type Modus Reported size (nm) 60.75 EV concentration Yes

	EV200092	5/5	Homo sapiens	Urine	(d)(U)C Filtration DG	Dong, Liang	2021	50%
Study summary Full title Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium All authors Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue Journal J Extracell Vesicles Abstract One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (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 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 (d)(U)C Filtration DG Protein markers EV: CD81/ Flotillin1 non-EV: None Proteomics no EV density (g/ml) 1.10-1.15 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 Between 10,000 g and 50,000 g Pelleting performed No Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 5% Highest density fraction 60% Total gradient volume, incl. sample (mL) 48 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 32 Ti Speed (g) 100000 Duration (min) 230 Fraction volume (mL) 4.75 Fraction processing Centrifugation Pelleting: volume per fraction 28 Pelleting: duration (min) 60 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 120000 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD81 Flow cytometry Type of Flow cytometry NanoFCM Hardware adaptation to ~100nm EV's Please refer to the following publication: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nan Calibration bead size 0.2 Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type NanoFCM Hardware adjustment Please refer to the following publication: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006. Calibration bead size 0.2

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EV-TRACK ID	EV200092
species	Homo sapiens
sample type	Urine
condition	Control condition
separation protocol	(d)(U)C Filtration	(d)(U)C Filtration Total Exosome Isolation	(d)(U)C Filtration Exodisc	(d)(U)C Filtration SEC and UF	(d)(U)C Filtration DG
Exp. nr.	1	2	3	4	5
EV-METRIC %	56	50	50	50	50