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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV180051	2/2	Rattus norvegicus	Blood plasma	(d)(U)C	Takov K	2018	66%
Study summary Full title Comparison of small extracellular vesicles isolated from plasma by ultracentrifugation or size-exclusion chromatography: yield, purity and functional potential. All authors Takov K, Yellon DM, Davidson SM Journal J Extracell Vesicles Abstract Interest in small extracellular vesicles (sEVs) as functional carriers of proteins and nucleic acids (show more...)Interest in small extracellular vesicles (sEVs) as functional carriers of proteins and nucleic acids is growing continuously. There are large numbers of sEVs in the blood, but lack of standardised methods for sEV isolation greatly limits our ability to study them. In this report, we use rat plasma to systematically compare two commonly used techniques for isolation of sEVs: ultracentrifugation (UC-sEVs) and size-exclusion chromatography (SEC-sEVs). SEC-sEVs had higher particle number, protein content, particle/protein ratios and sEV marker signal than UC-sEVs. However, SEC-sEVs also contained greater amounts of APOB⁺ lipoproteins and large quantities of non-sEV protein. sEV marker signal correlated very well with both particle number and protein content in UC-sEVs but not in all of the SEC-sEV fractions. Functionally, both UC-sEVs and SEC-sEVs isolates contained a variety of proangiogenic factors (with endothelin-1 being the most abundant) and stimulated migration of endothelial cells. However, there was no evident correlation between the promigratory potential and the quantity of sEVs added, indicating that non-vesicular co-isolates may contribute to the promigratory effects. Overall, our findings suggest that UC provides plasma sEVs of lower yields, but markedly higher purity compared to SEC. Furthermore, we show that the functional activity of sEVs can depend on the isolation method used and does not solely reflect the sEV quantity. These findings are of importance when working with sEVs isolated from plasma- or serum-containing conditioned medium. (hide) EV-METRIC 66% (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 small 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 Adj. k-factor 115.3 (pelleting) / 115.3 (washing) Protein markers EV: CD81/ HSP70/ Endothelin-1/ CD9 non-EV: ApoB Proteomics no Show all info Study aim Function, Technical analysis comparing/optimizing EV-related methods, Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type MLA-55 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 115.3 Wash: time (min) 70 Wash: Rotor Type MLA-55 Wash: speed (g) 100000 Wash: adjusted k-factor 115.3 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) 13.6-14.6 Western Blot Detected EV-associated proteins HSP70 ELISA Other 1 Protein array (ARY007, R&D Systems) Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 96.6 EV concentration Yes Particle yield 20000000000 EM EM-type Transmission-EM Image type Wide-field

	EV180051	1/2	Rattus norvegicus	Blood plasma	(d)(U)C qEV UF	Takov K	2018	62%
Study summary Full title Comparison of small extracellular vesicles isolated from plasma by ultracentrifugation or size-exclusion chromatography: yield, purity and functional potential. All authors Takov K, Yellon DM, Davidson SM Journal J Extracell Vesicles Abstract Interest in small extracellular vesicles (sEVs) as functional carriers of proteins and nucleic acids (show more...)Interest in small extracellular vesicles (sEVs) as functional carriers of proteins and nucleic acids is growing continuously. There are large numbers of sEVs in the blood, but lack of standardised methods for sEV isolation greatly limits our ability to study them. In this report, we use rat plasma to systematically compare two commonly used techniques for isolation of sEVs: ultracentrifugation (UC-sEVs) and size-exclusion chromatography (SEC-sEVs). SEC-sEVs had higher particle number, protein content, particle/protein ratios and sEV marker signal than UC-sEVs. However, SEC-sEVs also contained greater amounts of APOB⁺ lipoproteins and large quantities of non-sEV protein. sEV marker signal correlated very well with both particle number and protein content in UC-sEVs but not in all of the SEC-sEV fractions. Functionally, both UC-sEVs and SEC-sEVs isolates contained a variety of proangiogenic factors (with endothelin-1 being the most abundant) and stimulated migration of endothelial cells. However, there was no evident correlation between the promigratory potential and the quantity of sEVs added, indicating that non-vesicular co-isolates may contribute to the promigratory effects. Overall, our findings suggest that UC provides plasma sEVs of lower yields, but markedly higher purity compared to SEC. Furthermore, we show that the functional activity of sEVs can depend on the isolation method used and does not solely reflect the sEV quantity. These findings are of importance when working with sEVs isolated from plasma- or serum-containing conditioned medium. (hide) EV-METRIC 62% (89th 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 small 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 qEV UF Protein markers EV: CD81/ HSP70/ Endothelin-1/ CD9 non-EV: ApoB Proteomics no Show all info Study aim Function, Technical analysis comparing/optimizing EV-related methods, Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) 39-308 Western Blot Detected EV-associated proteins HSP70 ELISA Other 1 Protein array (ARY007, R&D Systems) Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 81.5 EV concentration Yes Particle yield 2000000000000 EM EM-type Transmission-EM Image type Wide-field

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EV-TRACK ID	EV180051
species	Rattus norvegicus
sample type	Blood plasma
condition	Control condition
separation protocol	(d)(U)C	(d)(U)C qEV UF
Exp. nr.	2	1
EV-METRIC %	66	62