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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140137	2/2	Homo sapiens	NAY	(d)(U)C Salting	Brownlee Z	2014	25%
Study summary Full title A novel salting-out procedure for the isolation of tumor-derived exosomes All authors Brownlee Z, Lynn KD, Thorpe PE, Schroit AJ Journal J Immunol Methods Abstract The last decade has seen an exponential growth in the number of exosome-related publications. Althou (show more...)The last decade has seen an exponential growth in the number of exosome-related publications. Although many of these studies have used exosomes from biological fluids (blood, and ascites or pleural effusions) the vast majority employed vesicles isolated from large volumes of tissue culture supernatants. While several techniques are available for their isolation, all require a significant reduction in volume to obtain sufficient concentrations for study. One approach is to concentrate the medium before proceeding with their isolation, however, these procedures are very time consuming and require specialized laboratory equipment. Here we provide a new and effective method for the isolation of tumor-derived exosomes based on charge neutralization with acetate. We show that titration of tissue culture supernatants with 0.1M acetate to pH4.75 results in immediate precipitation of virtually all the exosomes. The precipitated exosomes can be washed to remove residual media and are readily resolubilized upon resuspension in acetate-free buffer at neutral pH. This simple cost effective method significantly increases the yield of exosomes from an unlimited quantity of culture supernatants. Exosomes isolated by this technique are indistinguishable from exosomes recovered by direct ultracentrifugation. (hide) EV-METRIC 25% (64th 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 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 Salting Protein markers EV: Alix/ HSP70/ Annexin5 non-EV: Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Other Name other separation method Salting Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ HSP70/ Annexin5 ELISA Antibody details provided? No Detected EV-associated proteins Annexin5 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

	EV140137	1/2	Homo sapiens	NAY	(d)(U)C	Brownlee Z	2014	22%
Study summary Full title A novel salting-out procedure for the isolation of tumor-derived exosomes All authors Brownlee Z, Lynn KD, Thorpe PE, Schroit AJ Journal J Immunol Methods Abstract The last decade has seen an exponential growth in the number of exosome-related publications. Althou (show more...)The last decade has seen an exponential growth in the number of exosome-related publications. Although many of these studies have used exosomes from biological fluids (blood, and ascites or pleural effusions) the vast majority employed vesicles isolated from large volumes of tissue culture supernatants. While several techniques are available for their isolation, all require a significant reduction in volume to obtain sufficient concentrations for study. One approach is to concentrate the medium before proceeding with their isolation, however, these procedures are very time consuming and require specialized laboratory equipment. Here we provide a new and effective method for the isolation of tumor-derived exosomes based on charge neutralization with acetate. We show that titration of tissue culture supernatants with 0.1M acetate to pH4.75 results in immediate precipitation of virtually all the exosomes. The precipitated exosomes can be washed to remove residual media and are readily resolubilized upon resuspension in acetate-free buffer at neutral pH. This simple cost effective method significantly increases the yield of exosomes from an unlimited quantity of culture supernatants. Exosomes isolated by this technique are indistinguishable from exosomes recovered by direct ultracentrifugation. (hide) EV-METRIC 22% (59th 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 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 Protein markers EV: Alix/ HSP70/ Annexin5 non-EV: Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free 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) 60 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ HSP70/ Annexin5 ELISA Antibody details provided? No Detected EV-associated proteins Annexin5 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

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EV-TRACK ID	EV140137
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C Salting	(d)(U)C
Exp. nr.	2	1
EV-METRIC %	25	22