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Results list Most used separation protocols Top EV-enriched proteins

Details	EV-TRACK ID	Experiment nr.	Species	Sample type	separation protocol	First author	Year	EV-METRIC
	EV120004	1/1	Homo sapiens	Cell culture supernatant	DG (d)(U)C	de Jong OG	2012	67%
Study summary Full title Cellular stress conditions are reflected in the protein and RNA content of endothelial cell-derived exosomes All authors de Jong OG, Verhaar MC, Chen Y, Vader P, Gremmels H, Posthuma G, Schiffelers RM, Gucek M, van Balkom BW Journal J Extracell Vesicles Abstract BACKGROUND: The healthy vascular endothelium, which forms the barrier between blood and the surround (show more...)BACKGROUND: The healthy vascular endothelium, which forms the barrier between blood and the surrounding tissues, is known to efficiently respond to stress signals like hypoxia and inflammation by adaptation of cellular physiology and the secretion of (soluble) growth factors and cytokines. Exosomes are potent mediators of intercellular communication. Their content consists of RNA and proteins from the cell of origin, and thus depends on the condition of these cells at the time of exosome biogenesis. It has been suggested that exosomes protect their target cells from cellular stress through the transfer of RNA and proteins. We hypothesized that endothelium-derived exosomes are involved in the endothelial response to cellular stress, and that exosome RNA and protein content reflect the effects of cellular stress induced by hypoxia, inflammation or hyperglycemia. METHODS: We exposed cultured endothelial cells to different types of cellular stress (hypoxia, TNF-?-induced activation, high glucose and mannose concentrations) and compared mRNA and protein content of exosomes produced by these cells by microarray analysis and a quantitative proteomics approach. RESULTS: We identified 1,354 proteins and 1,992 mRNAs in endothelial cell-derived exosomes. Several proteins and mRNAs showed altered abundances after exposure of their producing cells to cellular stress, which were confirmed by immunoblot or qPCR analysis. CONCLUSION: Our data show that hypoxia and endothelial activation are reflected in RNA and protein exosome composition, and that exposure to high sugar concentrations alters exosome protein composition only to a minor extend, and does not affect exosome RNA composition. (hide) EV-METRIC 67% (93rd 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 exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + (d)(U)C Adj. k-factor 256 (pelleting) Protein markers EV: HSP70/ Beta-actin/ Flotilin1/ CD63/ ICAM1 non-EV: Proteomics yes EV density (g/ml) 1.09-1.12 Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method Differential ultracentrifugation centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 60 Pelleting: rotor type SW32;SW60 Pelleting: adjusted k-factor 256.0 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Bottom-up Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ Flotilin1/ HSP70/ Beta-actin/ ICAM1 ELISA Detected EV-associated proteins Beta-actin/ ICAM1 Characterization: Particle analysis NTA EM EM-type immune EM Proteïns CD63 Image type Wide-field

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