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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230602	3/3	Homo sapiens	Blood plasma	(d)(U)C	Benayas, Beatriz	2023	67%
Study summary Full title Optimization of extracellular vesicle isolation and their separation from lipoproteins by size exclusion chromatography All authors Beatriz Benayas, Joaquín Morales, Carolina Egea, Pilar Armisén, María Yáñez-Mó Journal J Extracell Biol Abstract Interest in the use of extracellular vesicles (EVs) as biomarkers of disease is rapidly growing. How (show more...)Interest in the use of extracellular vesicles (EVs) as biomarkers of disease is rapidly growing. However, one main unsolved issue in the EV field is finding a technique able to eliminate non-EV contaminants present in biofluid samples in a one-step isolation protocol. Due to the expansion and value of size exclusion chromatography (SEC) as one of the best EV isolation methods, we have tested several agarose resins with different agarose percentages, bead sizes and crosslinking features to optimize EV isolation. For this optimization of SEC, we first employed conditioned media from a melanoma cell culture, a simpler sample in comparison to biological fluids, but which also contains abundant contaminants such as soluble protein and lipoproteins (LPPs). The distinct agaroses and the combinations of resins with different agarose percentages in the same column were tested. Soluble protein, EVs and LPPs levels from the different eluted fractions were quantitated by immunodetection or absorbance measurements. Samples were also analysed by NTA and TEM to verify the yield and the LPP contamination. Different percentages of agarose resins (2%, 4% and 6%) yielded samples with increasing LPP contamination respectively, which was not improved in the columns that combined them. Crosslinking of the agarose did not affect EV isolation yield nor the LPP contamination. In contrast, reducing the bead size greatly improved EV purity. We thus selected 4% Rapid Run Fine agarose beads as the resin that more efficiently isolated EVs with almost no contamination of other particles. Using blood plasma samples, this resin also demonstrated an improved capacity in the isolation of EVs from LPPs in comparison to the agaroses most commonly used in the field and differential ultracentrifugation. (hide) EV-METRIC 67% (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 Protein markers EV: CD9/ CD81 non-EV: ApoB/ ApoE Proteomics no 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 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: rotor type AH 627 Pelleting: speed (g) 100000 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/ CD81 Not detected contaminants ApoB/ ApoE Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 120 EV concentration Yes Particle yield particles per milliliter of starting sample: 3.00E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230602	1/3	Homo sapiens	SK-MEL-147	(d)(U)C UF SEC (non-commercial)	Benayas, Beatriz	2023	63%
Study summary Full title Optimization of extracellular vesicle isolation and their separation from lipoproteins by size exclusion chromatography All authors Beatriz Benayas, Joaquín Morales, Carolina Egea, Pilar Armisén, María Yáñez-Mó Journal J Extracell Biol Abstract Interest in the use of extracellular vesicles (EVs) as biomarkers of disease is rapidly growing. How (show more...)Interest in the use of extracellular vesicles (EVs) as biomarkers of disease is rapidly growing. However, one main unsolved issue in the EV field is finding a technique able to eliminate non-EV contaminants present in biofluid samples in a one-step isolation protocol. Due to the expansion and value of size exclusion chromatography (SEC) as one of the best EV isolation methods, we have tested several agarose resins with different agarose percentages, bead sizes and crosslinking features to optimize EV isolation. For this optimization of SEC, we first employed conditioned media from a melanoma cell culture, a simpler sample in comparison to biological fluids, but which also contains abundant contaminants such as soluble protein and lipoproteins (LPPs). The distinct agaroses and the combinations of resins with different agarose percentages in the same column were tested. Soluble protein, EVs and LPPs levels from the different eluted fractions were quantitated by immunodetection or absorbance measurements. Samples were also analysed by NTA and TEM to verify the yield and the LPP contamination. Different percentages of agarose resins (2%, 4% and 6%) yielded samples with increasing LPP contamination respectively, which was not improved in the columns that combined them. Crosslinking of the agarose did not affect EV isolation yield nor the LPP contamination. In contrast, reducing the bead size greatly improved EV purity. We thus selected 4% Rapid Run Fine agarose beads as the resin that more efficiently isolated EVs with almost no contamination of other particles. Using blood plasma samples, this resin also demonstrated an improved capacity in the isolation of EVs from LPPs in comparison to the agaroses most commonly used in the field and differential ultracentrifugation. (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 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 Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD63/ CD81/ TSG101/ Syntenin non-EV: Calnexin/ VDAC/ ApoB Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SK-MEL-147 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) 95 Cell count 25000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Resins from Agarose Bead Technologies and Cytiva 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/ TSG101/ Syntenin Not detected contaminants Calnexin/ VDAC/ ApoB Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes Particle yield particles per milliliter of starting sample: 5.00E+09 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 110

	EV230602	2/3	Homo sapiens	Blood plasma	(d)(U)C SEC (non-commercial)	Benayas, Beatriz	2023	63%
Study summary Full title Optimization of extracellular vesicle isolation and their separation from lipoproteins by size exclusion chromatography All authors Beatriz Benayas, Joaquín Morales, Carolina Egea, Pilar Armisén, María Yáñez-Mó Journal J Extracell Biol Abstract Interest in the use of extracellular vesicles (EVs) as biomarkers of disease is rapidly growing. How (show more...)Interest in the use of extracellular vesicles (EVs) as biomarkers of disease is rapidly growing. However, one main unsolved issue in the EV field is finding a technique able to eliminate non-EV contaminants present in biofluid samples in a one-step isolation protocol. Due to the expansion and value of size exclusion chromatography (SEC) as one of the best EV isolation methods, we have tested several agarose resins with different agarose percentages, bead sizes and crosslinking features to optimize EV isolation. For this optimization of SEC, we first employed conditioned media from a melanoma cell culture, a simpler sample in comparison to biological fluids, but which also contains abundant contaminants such as soluble protein and lipoproteins (LPPs). The distinct agaroses and the combinations of resins with different agarose percentages in the same column were tested. Soluble protein, EVs and LPPs levels from the different eluted fractions were quantitated by immunodetection or absorbance measurements. Samples were also analysed by NTA and TEM to verify the yield and the LPP contamination. Different percentages of agarose resins (2%, 4% and 6%) yielded samples with increasing LPP contamination respectively, which was not improved in the columns that combined them. Crosslinking of the agarose did not affect EV isolation yield nor the LPP contamination. In contrast, reducing the bead size greatly improved EV purity. We thus selected 4% Rapid Run Fine agarose beads as the resin that more efficiently isolated EVs with almost no contamination of other particles. Using blood plasma samples, this resin also demonstrated an improved capacity in the isolation of EVs from LPPs in comparison to the agaroses most commonly used in the field and differential ultracentrifugation. (hide) EV-METRIC 63% (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 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 Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD81 non-EV: ApoB/ ApoE Proteomics no 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 Pelleting performed No Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Resins from Agarose Bead Technologies 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/ CD81 Not detected contaminants ApoB/ ApoE Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 100 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.00E+11 EM EM-type Transmission-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV230602
species	Homo sapiens
sample type	Blood plasma	Cell culture	Blood plasma
cell type	NA	SK-MEL-147	NA
medium	NA	EV-depleted medium	NA
condition	Control condition	Control condition	Control condition
separation protocol	dUC	dUC/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)	dUC/ Size-exclusion chromatography (non-commercial)
Exp. nr.	3	1	2
EV-METRIC %	67	63	63