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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	separation protocol	First author	Year	EV-METRIC
	EV140001	2/4	Homo sapiens	Cell culture supernatant	DG UF Filtration dUC	Van Deun J	2014	88%
Study summary Full title The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling All authors Van Deun J, Mestdagh P, Sormunen R, Cocquyt V, Vermaelen K, Vandesompele J, Bracke M, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer an (show more...)Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer and their use as biomarkers for diagnosis, prognosis, drug response and recurrence, there is no consensus on dependable isolation protocols. We provide a comparative evaluation of 4 exosome isolation protocols for their usability, yield and purity, and their impact on downstream omics approaches for biomarker discovery. OptiPrep density gradient centrifugation outperforms ultracentrifugation and ExoQuick and Total Exosome Isolation precipitation in terms of purity, as illustrated by the highest number of CD63-positive nanovesicles, the highest enrichment in exosomal marker proteins and a lack of contaminating proteins such as extracellular Argonaute-2 complexes. The purest exosome fractions reveal a unique mRNA profile enriched for translation, ribosome, mitochondrion and nuclear lumen function. Our results demonstrate that implementation of high purification techniques is a prerequisite to obtain reliable omics data and identify exosome-specific functions and biomarkers. (hide) EV-METRIC 88% (98th 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 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 DNF Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + UF + Filtration + dUC Protein markers EV: Alix/ HSP70/ TSG101/ HSP90/ CD63 non-EV: Cell organelle protein/ Ago2 Proteomics no EV density (g/ml) 1.094 Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Density gradient Density medium Iodixanol Lowest density fraction 5 Highest density fraction 40 Orientation Top-down Rotor type SW32.1 Speed (g) 100000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63/ HSP90/ HSP70/ TSG101 Detected contaminants Cell organelle protein/ Ago2 Characterization: Particle analysis NTA EM EM-type immune EM Proteïns CD63 Image type Wide-field

	EV140001	1/4	Homo sapiens	Cell culture supernatant	dUC Filtration UF	Van Deun J	2014	67%
Study summary Full title The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling All authors Van Deun J, Mestdagh P, Sormunen R, Cocquyt V, Vermaelen K, Vandesompele J, Bracke M, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer an (show more...)Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer and their use as biomarkers for diagnosis, prognosis, drug response and recurrence, there is no consensus on dependable isolation protocols. We provide a comparative evaluation of 4 exosome isolation protocols for their usability, yield and purity, and their impact on downstream omics approaches for biomarker discovery. OptiPrep density gradient centrifugation outperforms ultracentrifugation and ExoQuick and Total Exosome Isolation precipitation in terms of purity, as illustrated by the highest number of CD63-positive nanovesicles, the highest enrichment in exosomal marker proteins and a lack of contaminating proteins such as extracellular Argonaute-2 complexes. The purest exosome fractions reveal a unique mRNA profile enriched for translation, ribosome, mitochondrion and nuclear lumen function. Our results demonstrate that implementation of high purification techniques is a prerequisite to obtain reliable omics data and identify exosome-specific functions and biomarkers. (hide) EV-METRIC 67% (94th 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 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 DNF Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Filtration + UF Adj. k-factor 138.6 (pelleting) Protein markers EV: Alix/ HSP70/ TSG101/ HSP90/ CD63 non-EV: Cell organelle protein/ Ago2 Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 100,000 g and 150,000 g Pelleting: time(min) 180 Pelleting: rotor type SW55 Pelleting: adjusted k-factor 138.6 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63/ HSP90/ HSP70/ TSG101 Detected contaminants Cell organelle protein/ Ago2 Characterization: Particle analysis NTA EM EM-type immune EM Proteïns CD63 Image type Wide-field

	EV140001	3/4	Homo sapiens	Cell culture supernatant	Total Exosome Isolation UF Filtration dUC	Van Deun J	2014	63%
Study summary Full title The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling All authors Van Deun J, Mestdagh P, Sormunen R, Cocquyt V, Vermaelen K, Vandesompele J, Bracke M, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer an (show more...)Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer and their use as biomarkers for diagnosis, prognosis, drug response and recurrence, there is no consensus on dependable isolation protocols. We provide a comparative evaluation of 4 exosome isolation protocols for their usability, yield and purity, and their impact on downstream omics approaches for biomarker discovery. OptiPrep density gradient centrifugation outperforms ultracentrifugation and ExoQuick and Total Exosome Isolation precipitation in terms of purity, as illustrated by the highest number of CD63-positive nanovesicles, the highest enrichment in exosomal marker proteins and a lack of contaminating proteins such as extracellular Argonaute-2 complexes. The purest exosome fractions reveal a unique mRNA profile enriched for translation, ribosome, mitochondrion and nuclear lumen function. Our results demonstrate that implementation of high purification techniques is a prerequisite to obtain reliable omics data and identify exosome-specific functions and biomarkers. (hide) EV-METRIC 63% (92nd 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 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 DNF Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography Total Exosome Isolation + + UF + Filtration + dUC Adj. k-factor 0 (pelleting) Protein markers EV: Alix/ HSP70/ TSG101/ HSP90/ CD63 non-EV: Cell organelle protein/ Ago2 Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting: adjusted k-factor NA Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Commercial kit Total Exosome Isolation Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63/ HSP90/ HSP70/ TSG101 Detected contaminants Cell organelle protein/ Ago2 Characterization: Particle analysis NTA EM EM-type immune EM Proteïns CD63 Image type Close-up

	EV140001	4/4	Homo sapiens	Cell culture supernatant	UF Filtration ExoQuick dUC	Van Deun J	2014	63%
Study summary Full title The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling All authors Van Deun J, Mestdagh P, Sormunen R, Cocquyt V, Vermaelen K, Vandesompele J, Bracke M, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer an (show more...)Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer and their use as biomarkers for diagnosis, prognosis, drug response and recurrence, there is no consensus on dependable isolation protocols. We provide a comparative evaluation of 4 exosome isolation protocols for their usability, yield and purity, and their impact on downstream omics approaches for biomarker discovery. OptiPrep density gradient centrifugation outperforms ultracentrifugation and ExoQuick and Total Exosome Isolation precipitation in terms of purity, as illustrated by the highest number of CD63-positive nanovesicles, the highest enrichment in exosomal marker proteins and a lack of contaminating proteins such as extracellular Argonaute-2 complexes. The purest exosome fractions reveal a unique mRNA profile enriched for translation, ribosome, mitochondrion and nuclear lumen function. Our results demonstrate that implementation of high purification techniques is a prerequisite to obtain reliable omics data and identify exosome-specific functions and biomarkers. (hide) EV-METRIC 63% (92nd 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 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 DNF Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography UF + Filtration + ExoQuick + dUC Protein markers EV: Alix/ HSP70/ TSG101/ HSP90/ CD63 non-EV: Cell organelle protein/ Ago2 Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Commercial kit ExoQuick Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63/ HSP90/ HSP70/ TSG101 Detected contaminants Cell organelle protein/ Ago2 Characterization: Particle analysis NTA EM EM-type immune EM Proteïns CD63 Image type Close-up

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EV-TRACK ID	EV140001
species	Homo sapiens
sample type	Cell culture
separation protocol	DG UF Filtration dUC	dUC Filtration UF	Total Exosome Isolation UF Filtration dUC	UF Filtration ExoQuick dUC
Exp. nr.	2	1	3	4
EV-METRIC %	88	67	63	63