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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV180039	1/2	Bos taurus	Non-conditioned medium	DG (d)(U)C	Driedonks, Tom	2018	42%
Study summary Full title Technical approaches to reduce interference of Fetal calf serum derived RNA in the analysis of extracellular vesicle RNA from cultured cells All authors Tom A P Driedonks, Maarten K Nijen Twilhaar, Esther N M Nolte-'t Hoen Journal J Extracell Vesicles Abstract Foetal calf serum (FCS) is a common supplement of cell culture medium and a known source of contamin (show more...)Foetal calf serum (FCS) is a common supplement of cell culture medium and a known source of contaminating extracellular vesicles (EV) containing RNA. Because of a high degree of sequence similarity among homologous non-coding RNAs of mammalian species, residual FCS-RNA in culture medium may interfere in the analysis of EV-RNA released by cultured cells. Recently, doubts have been raised as to whether commonly used protocols for depletion of FCS-EV efficiently remove FCS-RNA. Moreover, technical details in FCS-EV depletion protocols are known to vary between labs, which may lead to inter-study differences in contaminating FCS-RNA levels. Here, we investigated how technical modifications of EV-depletion protocols affect the efficiency with which bovine RNAs are depleted from FCS, and determined the contribution of contaminating bovine RNA to EV-RNA purified from cell cultures. Our data show differences in depletion efficiency between and within various classes of small non-coding RNA. Importantly, we demonstrate that variations in FCS-EV depletion protocols affect both the quantity and type of residual FCS-RNAs in EV-depleted medium. By using optimised FCS-EV depletion protocols combined with methods for high-grade purification of EV the levels of contaminating bovine RNA in EV populations isolated from cell culture medium can be reduced. With illustrative datasets we also demonstrate that the abundance of a specific RNA in cell culture EV can only be determined if measured relative to background levels of this RNA in medium control samples. These data highlight the need for optimisation and validation of existing and novel FCS-EV depletion methods and urge for accurate descriptions of these methods in publications to increase experimental reproducibility. (hide) EV-METRIC 42% (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 Non-conditioned medium 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 DG (d)(U)C Adj. k-factor 253.9 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type Non-conditioned medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Sample volume (mL) 0.05 Orientation Bottom-up (sample migrates upwards) Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 900 - 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 6 Pelleting: duration (min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 192000 Pelleting: adjusted k-factor 144.0 Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx Hardware adjustment see van der Vlist et al. 2012 Nature Protocols;and Nolte-'t Hoen 2012 Nanomedicine Calibration bead size 0.1,0.2 EV concentration Yes

	EV180039	2/2	Homo sapiens	HEK293T	DG (d)(U)C	Driedonks, Tom	2018	42%
Study summary Full title Technical approaches to reduce interference of Fetal calf serum derived RNA in the analysis of extracellular vesicle RNA from cultured cells All authors Tom A P Driedonks, Maarten K Nijen Twilhaar, Esther N M Nolte-'t Hoen Journal J Extracell Vesicles Abstract Foetal calf serum (FCS) is a common supplement of cell culture medium and a known source of contamin (show more...)Foetal calf serum (FCS) is a common supplement of cell culture medium and a known source of contaminating extracellular vesicles (EV) containing RNA. Because of a high degree of sequence similarity among homologous non-coding RNAs of mammalian species, residual FCS-RNA in culture medium may interfere in the analysis of EV-RNA released by cultured cells. Recently, doubts have been raised as to whether commonly used protocols for depletion of FCS-EV efficiently remove FCS-RNA. Moreover, technical details in FCS-EV depletion protocols are known to vary between labs, which may lead to inter-study differences in contaminating FCS-RNA levels. Here, we investigated how technical modifications of EV-depletion protocols affect the efficiency with which bovine RNAs are depleted from FCS, and determined the contribution of contaminating bovine RNA to EV-RNA purified from cell cultures. Our data show differences in depletion efficiency between and within various classes of small non-coding RNA. Importantly, we demonstrate that variations in FCS-EV depletion protocols affect both the quantity and type of residual FCS-RNAs in EV-depleted medium. By using optimised FCS-EV depletion protocols combined with methods for high-grade purification of EV the levels of contaminating bovine RNA in EV populations isolated from cell culture medium can be reduced. With illustrative datasets we also demonstrate that the abundance of a specific RNA in cell culture EV can only be determined if measured relative to background levels of this RNA in medium control samples. These data highlight the need for optimisation and validation of existing and novel FCS-EV depletion methods and urge for accurate descriptions of these methods in publications to increase experimental reproducibility. (hide) EV-METRIC 42% (80th 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 DG (d)(U)C Adj. k-factor 253.9 (pelleting) Protein markers EV: None non-EV: None 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 HEK293T EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) NA Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Sample volume (mL) 0.05 Orientation Bottom-up (sample migrates upwards) Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 900 - 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 6 Pelleting: duration (min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 192000 Pelleting: adjusted k-factor 144.0 Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx Hardware adjustment see van der Vlist et al. 2012 Nature Protocols;and Nolte-'t Hoen 2012 Nanomedicine Calibration bead size 0.1,0.2 EV concentration Yes

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EV-TRACK ID	EV180039
species	Bos taurus	Homo sapiens
sample type	Non-conditioned medium	Cell culture
cell type	NA	HEK293T
medium	NA	EV-depleted serum
condition	Control condition	Control condition
separation protocol	DG (d)(U)C	DG (d)(U)C
Exp. nr.	1	2
EV-METRIC %	42	42