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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200025	3/4	Homo sapiens	Blood plasma	UF SEC (non-commercial) cation-exchange chromatography	Van Deun J	2020	63%
Study summary Full title Integrated Dual-Mode Chromatography to Enrich Extracellular Vesicles from Plasma. All authors Van Deun J, Jo A, Li H, Lin HY, Weissleder R, Im H, Lee H Journal Adv Biosyst Abstract Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzin (show more...)Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzing EVs molecularly. Plasma lipoprotein particles (LPPs) are a significant confounding factor as they outnumber EVs >10 -fold. Given their overlap in size, LPPs cannot be completely removed using standard size-exclusion chromatography. Density-based separation of LPPs can be applied but is impractical for routine use in clinical research and practice. Here a new separation approach, known as dual-mode chromatography (DMC), capable of enriching plasma EVs, and depleting LPPs is reported. DMC conveniently integrates two orthogonal separation steps in a single column device: i) size exclusion to remove high-density lipoproteins (HDLs) that are smaller than EVs/ and ii) cation exchange to clear positively charged ApoB100-containing LPPs, mostly (very) low-density lipoproteins (V)LDLs, from negatively charged EVs. The strategy enables DMC to deplete most LPPs (>97% of HDLs and >99% of (V)LDLs) from human plasma, while retaining EVs (>30% of input). Furthermore, the two-in-one operation is fast (15 min per sample) and equipment-free. With abundant LPPs removed, DMC-prepared samples facilitate EV identification in imaging analyses and improve the accuracy for EV protein analysis. (hide) EV-METRIC 63% (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 Spiked with ES2 EVs 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 Size-exclusion chromatography (non-commercial) cation-exchange chromatography Protein markers EV: CD63 non-EV: ApoA1/ ApoB100 Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Sepharose CL-4B Other Name other separation method cation-exchange chromatography Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63 Detected contaminants ApoA1 ELISA Detected contaminants ApoB100 Characterization: Lipid analysis No Characterization: Particle analysis NTA EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200025	4/4	Homo sapiens	Blood plasma	UF SEC (non-commercial)	Van Deun J	2020	63%
Study summary Full title Integrated Dual-Mode Chromatography to Enrich Extracellular Vesicles from Plasma. All authors Van Deun J, Jo A, Li H, Lin HY, Weissleder R, Im H, Lee H Journal Adv Biosyst Abstract Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzin (show more...)Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzing EVs molecularly. Plasma lipoprotein particles (LPPs) are a significant confounding factor as they outnumber EVs >10 -fold. Given their overlap in size, LPPs cannot be completely removed using standard size-exclusion chromatography. Density-based separation of LPPs can be applied but is impractical for routine use in clinical research and practice. Here a new separation approach, known as dual-mode chromatography (DMC), capable of enriching plasma EVs, and depleting LPPs is reported. DMC conveniently integrates two orthogonal separation steps in a single column device: i) size exclusion to remove high-density lipoproteins (HDLs) that are smaller than EVs/ and ii) cation exchange to clear positively charged ApoB100-containing LPPs, mostly (very) low-density lipoproteins (V)LDLs, from negatively charged EVs. The strategy enables DMC to deplete most LPPs (>97% of HDLs and >99% of (V)LDLs) from human plasma, while retaining EVs (>30% of input). Furthermore, the two-in-one operation is fast (15 min per sample) and equipment-free. With abundant LPPs removed, DMC-prepared samples facilitate EV identification in imaging analyses and improve the accuracy for EV protein analysis. (hide) EV-METRIC 63% (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 Spiked with ES2 EVs 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 Size-exclusion chromatography (non-commercial) Protein markers EV: CD63 non-EV: ApoA1/ ApoB100 Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Sepharose CL-4B Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63 Detected contaminants ApoA1 ELISA Detected contaminants ApoB100 Characterization: Lipid analysis No Characterization: Particle analysis NTA EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200025	1/4	Homo sapiens	ES2	(d)(U)C DG UF	Van Deun J	2020	57%
Study summary Full title Integrated Dual-Mode Chromatography to Enrich Extracellular Vesicles from Plasma. All authors Van Deun J, Jo A, Li H, Lin HY, Weissleder R, Im H, Lee H Journal Adv Biosyst Abstract Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzin (show more...)Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzing EVs molecularly. Plasma lipoprotein particles (LPPs) are a significant confounding factor as they outnumber EVs >10 -fold. Given their overlap in size, LPPs cannot be completely removed using standard size-exclusion chromatography. Density-based separation of LPPs can be applied but is impractical for routine use in clinical research and practice. Here a new separation approach, known as dual-mode chromatography (DMC), capable of enriching plasma EVs, and depleting LPPs is reported. DMC conveniently integrates two orthogonal separation steps in a single column device: i) size exclusion to remove high-density lipoproteins (HDLs) that are smaller than EVs/ and ii) cation exchange to clear positively charged ApoB100-containing LPPs, mostly (very) low-density lipoproteins (V)LDLs, from negatively charged EVs. The strategy enables DMC to deplete most LPPs (>97% of HDLs and >99% of (V)LDLs) from human plasma, while retaining EVs (>30% of input). Furthermore, the two-in-one operation is fast (15 min per sample) and equipment-free. With abundant LPPs removed, DMC-prepared samples facilitate EV identification in imaging analyses and improve the accuracy for EV protein analysis. (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 Density gradient Ultrafiltration Protein markers EV: CD9/ CD63/ CD81 non-EV: None Proteomics no EV density (g/ml) 1.1 Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells ES2 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 17 Sample volume (mL) 1 Orientation Top-down Rotor type SW27.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Resin type Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry specific beads Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV200025	2/4	Homo sapiens	Gli36 EGFRvIII	(d)(U)C DG UF	Van Deun J	2020	57%
Study summary Full title Integrated Dual-Mode Chromatography to Enrich Extracellular Vesicles from Plasma. All authors Van Deun J, Jo A, Li H, Lin HY, Weissleder R, Im H, Lee H Journal Adv Biosyst Abstract Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzin (show more...)Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzing EVs molecularly. Plasma lipoprotein particles (LPPs) are a significant confounding factor as they outnumber EVs >10 -fold. Given their overlap in size, LPPs cannot be completely removed using standard size-exclusion chromatography. Density-based separation of LPPs can be applied but is impractical for routine use in clinical research and practice. Here a new separation approach, known as dual-mode chromatography (DMC), capable of enriching plasma EVs, and depleting LPPs is reported. DMC conveniently integrates two orthogonal separation steps in a single column device: i) size exclusion to remove high-density lipoproteins (HDLs) that are smaller than EVs/ and ii) cation exchange to clear positively charged ApoB100-containing LPPs, mostly (very) low-density lipoproteins (V)LDLs, from negatively charged EVs. The strategy enables DMC to deplete most LPPs (>97% of HDLs and >99% of (V)LDLs) from human plasma, while retaining EVs (>30% of input). Furthermore, the two-in-one operation is fast (15 min per sample) and equipment-free. With abundant LPPs removed, DMC-prepared samples facilitate EV identification in imaging analyses and improve the accuracy for EV protein analysis. (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 Density gradient Ultrafiltration Protein markers EV: CD9/ CD63/ CD81 non-EV: None Proteomics no EV density (g/ml) 1.1 Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Gli36 EGFRvIII EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 17 Sample volume (mL) 1 Orientation Top-down Rotor type SW27.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Resin type Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry specific beads Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV200025
species	Homo sapiens
sample type	Blood plasma	Blood plasma	Cell culture	Cell culture
cell type	NA	NA	ES2	Gli36 EGFRvIII
medium	NA	NA	EV-depleted medium	EV-depleted medium
condition	Spiked with ES2 EVs	Spiked with ES2 EVs	Control condition	Control condition
separation protocol	Ultrafiltration/ Size-exclusion chromatography (non-commercial)/ cation-exchange chromatography	Ultrafiltration/ Size-exclusion chromatography (non-commercial)	dUC/ Density gradient/ Ultrafiltration	dUC/ Density gradient/ Ultrafiltration
Exp. nr.	3	4	1	2
EV-METRIC %	63	63	57	57