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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
	EV150106	1/1	Homo sapiens	Cell culture supernatant	DG (d)(U)C	van Balkom BW	2015	56%
Study summary Full title Quantitative and qualitative analysis of small RNAs in human endothelial cells and exosomes provides insights into localized RNA processing, degradation and sorting. All authors van Balkom BW, Eisele AS, Pegtel DM, Bervoets S, Verhaar MC. Journal J Extracell Vesicles Abstract Exosomes are small vesicles that mediate cell-cell communication. They contain proteins, lipids and (show more...)Exosomes are small vesicles that mediate cell-cell communication. They contain proteins, lipids and RNA, and evidence is accumulating that these molecules are specifically sorted for release via exosomes. We recently showed that endothelial-cell-produced exosomes promote angiogenesis in vivo in a small RNA-dependent manner. Recent deep sequencing studies in exosomes from lymphocytic origin revealed a broad spectrum of small RNAs. However, selective depletion or incorporation of small RNA species into endothelial exosomes has not been studied extensively. With next generation sequencing, we identified all known non-coding RNA classes, including microRNAs (miRNAs), small nucleolar RNAs, yRNAs, vault RNAs, 5p and 3p fragments of miRNAs and miRNA-like fragments. In addition, we mapped many fragments of messenger RNAs (mRNAs) and mitochondrial RNAs (mtRNAs). The distribution of small RNAs in exosomes revealed a considerable overlap with the distribution in the producing cells. However, we identified a remarkable enrichment of yRNA fragments and mRNA degradation products in exosomes consistent with yRNAs having a role in degradation of structured and misfolded RNAs in close proximity to endosomes. We propose that endothelial endosomes selectively sequester cytoplasmic RNA-degrading machineries taking part in gene regulation. The release of these regulatory RNAs via exosomes may have implications for endothelial cell-cell communication. (hide) EV-METRIC 56% (85th 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 Cell Name HMEC-1 Sample origin Control condition Focus vesicles exosome 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 IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: GAPDH/ Flotillin1/ CD9/ non-EV: Histone H2A.X/ Lamin A / C/ ATP5A/ Tom20 Proteomics no EV density (g/ml) 1.07 - 1.12 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells HMEC-1 EV-harvesting Medium EV-depleted medium Preparation of EDS 1h at 200000g 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 Not specified Pelleting: speed (g) 100000 Wash: time (min) 60 Wash: Rotor Type Not specified Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Density medium Sucrose Type Continuous Lowest density fraction 0.25M Highest density fraction 2.0M Sample volume (mL) 0.25 Orientation Bottom-up Rotor type Not specified Speed (g) 190000 Duration (min) 960 Fraction volume (mL) 0.4 Fraction processing None Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins GAPDH/ Flotillin1/ CD9 Not detected contaminants Lamin A/C/ Histone H2A.X/ ATP5A/ Tom20 EM EM-type Transmission-EM Image type Wide-field

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