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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV120111	1/3	Bos bovis	Milk	(d)(U)C DG UF	Yamada T	2012	33%
Study summary Full title Comparison of methods for isolating exosomes from bovine milk All authors Yamada T, Inoshima Y, Matsuda T, Ishiguro N Journal J Vet Med Sci Abstract Four methods were evaluated for isolating exosomes from bovine milk: (1) ExoQuick precipitation, (2) (show more...)Four methods were evaluated for isolating exosomes from bovine milk: (1) ExoQuick precipitation, (2) ultracentrifugation with ExoQuick precipitation, (3) ultracentrifugation with density gradient centrifugation, and (4) human milk exosome isolation. Methods 1 and 4 failed due to differences between bovine and human milk. Exosomes were efficiently isolated by ultracentrifugation with either ExoQuick precipitation (method 2) or density gradient centrifugation (method 3). The highest yield of exosomes was achieved using ultracentrifugation with ExoQuick precipitation, whereas higher quality exosome isolation with intact morphological structures was achieved by ultracentrifugation with density gradient centrifugation. (hide) EV-METRIC 33% (53rd 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 Milk Sample origin NAY Focus vesicles exosomes 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 DG UF Adj. k-factor 255.8 (pelleting) Protein markers EV: CD9/ MFGE8 non-EV: Proteomics no EV density (g/ml) 1.1-1.19 Show all info Study aim Technical Sample Species Bos bovis Sample Type Milk 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 Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW41 Pelleting: adjusted k-factor 255.8 Wash: volume per pellet (ml) 10 Density gradient Only used for validation of main results Yes Lowest density fraction 10 Highest density fraction 40 Orientation Top-down Rotor type MLA55 Speed (g) 100000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ MFGE8 ELISA Antibody details provided? No Detected EV-associated proteins MFGE8 Characterization: Particle analysis EM EM-type transmission EM

	EV120111	2/3	Bos bovis	Milk	(d)(U)C ExoQuick	Yamada T	2012	0%
Study summary Full title Comparison of methods for isolating exosomes from bovine milk All authors Yamada T, Inoshima Y, Matsuda T, Ishiguro N Journal J Vet Med Sci Abstract Four methods were evaluated for isolating exosomes from bovine milk: (1) ExoQuick precipitation, (2) (show more...)Four methods were evaluated for isolating exosomes from bovine milk: (1) ExoQuick precipitation, (2) ultracentrifugation with ExoQuick precipitation, (3) ultracentrifugation with density gradient centrifugation, and (4) human milk exosome isolation. Methods 1 and 4 failed due to differences between bovine and human milk. Exosomes were efficiently isolated by ultracentrifugation with either ExoQuick precipitation (method 2) or density gradient centrifugation (method 3). The highest yield of exosomes was achieved using ultracentrifugation with ExoQuick precipitation, whereas higher quality exosome isolation with intact morphological structures was achieved by ultracentrifugation with density gradient centrifugation. (hide) EV-METRIC 0% (median: 33% 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 Milk Sample origin NAY Focus vesicles exosomes 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 ExoQuick Protein markers EV: non-EV: Proteomics no Show all info Study aim Technical Sample Species Bos bovis Sample Type Milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Particle analysis None

	EV120111	3/3	Bos bovis	Milk	(d)(U)C ExoQuick Filtration	Yamada T	2012	0%
Study summary Full title Comparison of methods for isolating exosomes from bovine milk All authors Yamada T, Inoshima Y, Matsuda T, Ishiguro N Journal J Vet Med Sci Abstract Four methods were evaluated for isolating exosomes from bovine milk: (1) ExoQuick precipitation, (2) (show more...)Four methods were evaluated for isolating exosomes from bovine milk: (1) ExoQuick precipitation, (2) ultracentrifugation with ExoQuick precipitation, (3) ultracentrifugation with density gradient centrifugation, and (4) human milk exosome isolation. Methods 1 and 4 failed due to differences between bovine and human milk. Exosomes were efficiently isolated by ultracentrifugation with either ExoQuick precipitation (method 2) or density gradient centrifugation (method 3). The highest yield of exosomes was achieved using ultracentrifugation with ExoQuick precipitation, whereas higher quality exosome isolation with intact morphological structures was achieved by ultracentrifugation with density gradient centrifugation. (hide) EV-METRIC 0% (median: 33% 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 Milk Sample origin NAY Focus vesicles exosomes 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 ExoQuick Filtration Protein markers EV: CD9/ MFGE8 non-EV: Proteomics no Show all info Study aim Technical Sample Species Bos bovis Sample Type Milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed No Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ MFGE8 ELISA Antibody details provided? No Detected EV-associated proteins MFGE8 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

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EV-TRACK ID	EV120111
species	Bos bovis
sample type	Milk
condition	NAY
separation protocol	(d)(U)C DG UF	(d)(U)C ExoQuick	(d)(U)C ExoQuick Filtration
Exp. nr.	1	2	3
EV-METRIC %	33	0	0