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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200013	1/2	Bos taurus	Bovine ampulla oviductal fluid	DG qEV	Asaadi, Anise	2021	75%
Study summary Full title Extracellular Vesicles from Follicular and Ampullary Fluid Isolated by Density Gradient Ultracentrifugation Improve Bovine Embryo Development and Quality All authors Anise Asaadi, Nima Azari Dolatabad, Hadi Atashi, Annelies Raes, Petra Van Damme, Michael Hoelker, An Hendrix, Osvaldo Bogado Pascottini, Ann Van Soom, Mojtaba Kafi, Krishna Chaitanya Pavani Journal Int J Biol Sci Abstract Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AO (show more...)Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AOF), using different isolation methods. However, it is not clear whether different purification methods can affect the functionality of resulting EVs. Here, we compared two methods (OptiPrep™ density gradient ultracentrifugation (ODG UC) and single-step size exclusion chromatography (SEC) (qEV IZON™ single column)) for the isolation of EVs from bovine FF and AOF. Additionally, we evaluated whether the addition of EVs derived either by ODG UC or SEC from FF or AOF during oocyte maturation would yield extra benefits for embryo developmental competence. The characterization of EVs isolated using ODG UC or SEC from FF and AOF did not show any differences in terms of EV sizes (40-400 nm) and concentrations (2.4 ± 0.2 × 1012-1.8 ± 0.2 × 1013 particles/mL). Blastocyst yield and quality was higher in groups supplemented with EVs isolated from FF and AOF by ODG UC, with higher total cell numbers and a lower apoptotic cell ratio compared with the other groups (p < 0.05). Supplementing in vitro maturation media with EVs derived by ODG UC from AOF was beneficial for bovine embryo development and quality. (hide) EV-METRIC 75% (50th 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 Bovine ampulla oviductal fluid 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 Density gradient Commercial method Protein markers EV: TSG101/ CD63/ CD9 non-EV: None Proteomics no EV density (g/ml) 1.1 Show all info Study aim Function Sample Species Bos taurus Sample Type Bovine ampulla oviductal fluid Separation Method Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.8 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 14 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Commercial kit qEV Characterization: Protein analysis PMID previous EV protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63/ TSG101 Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Extra particle analysis NTA Report type Mean Reported size (nm) 165 +/- 5.8 nm EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1.80E+12 EM EM-type Transmission-EM Image type Close-up

	EV200013	2/2	Bos taurus	Bovine ovarian follicular fluid	DG qEV	Asaadi, Anise	2021	75%
Study summary Full title Extracellular Vesicles from Follicular and Ampullary Fluid Isolated by Density Gradient Ultracentrifugation Improve Bovine Embryo Development and Quality All authors Anise Asaadi, Nima Azari Dolatabad, Hadi Atashi, Annelies Raes, Petra Van Damme, Michael Hoelker, An Hendrix, Osvaldo Bogado Pascottini, Ann Van Soom, Mojtaba Kafi, Krishna Chaitanya Pavani Journal Int J Biol Sci Abstract Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AO (show more...)Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AOF), using different isolation methods. However, it is not clear whether different purification methods can affect the functionality of resulting EVs. Here, we compared two methods (OptiPrep™ density gradient ultracentrifugation (ODG UC) and single-step size exclusion chromatography (SEC) (qEV IZON™ single column)) for the isolation of EVs from bovine FF and AOF. Additionally, we evaluated whether the addition of EVs derived either by ODG UC or SEC from FF or AOF during oocyte maturation would yield extra benefits for embryo developmental competence. The characterization of EVs isolated using ODG UC or SEC from FF and AOF did not show any differences in terms of EV sizes (40-400 nm) and concentrations (2.4 ± 0.2 × 1012-1.8 ± 0.2 × 1013 particles/mL). Blastocyst yield and quality was higher in groups supplemented with EVs isolated from FF and AOF by ODG UC, with higher total cell numbers and a lower apoptotic cell ratio compared with the other groups (p < 0.05). Supplementing in vitro maturation media with EVs derived by ODG UC from AOF was beneficial for bovine embryo development and quality. (hide) EV-METRIC 75% (50th 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 Bovine ovarian follicular fluid 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 Density gradient Commercial method Protein markers EV: TSG101/ CD63/ CD9 non-EV: None Proteomics no EV density (g/ml) 1.1 Show all info Study aim Function Sample Species Bos taurus Sample Type Bovine ovarian follicular fluid Separation Method Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.8 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 14 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Commercial kit qEV Characterization: Protein analysis PMID previous EV protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63/ TSG101 Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Extra particle analysis NTA Report type Mean Reported size (nm) 166.9 +/- 9.7 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 6.44E+12 EM EM-type Transmission-EM Image type Close-up

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EV-TRACK ID	EV200013
species	Bos taurus
sample type	Bovine ampulla oviductal fluid	Bovine ovarian follicular fluid
condition	Control condition	Control condition
separation protocol	Density gradient qEV	Density gradient qEV
Exp. nr.	1	2
EV-METRIC %	75	75