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Results list Most used isolation protocols Top EV-enriched proteins

Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Isolation protocol	First author	Year	EV-METRIC
	EV110001	1/1	Homo sapiens	Cell culture supernatant	dUC Sucrose-DG UF	Atay S	2011	78%
Study summary Full title Morphologic and proteomic characterization of exosomes released by cultured extravillous trophoblast cells All authors Atay S, Gercel-Taylor C, Kesimer M, Taylor DD Journal Exp Cell Res Abstract Exosomes represent an important intercellular communication vehicle, mediating events essential for (show more...)Exosomes represent an important intercellular communication vehicle, mediating events essential for the decidual microenvironment. While we have demonstrated exosome induction of pro-inflammatory cytokines, to date, no extensive characterization of trophoblast-derived exosomes has been provided. Our objective was to provide a morphologic and proteomic characterization of these exosomes. Exosomes were isolated from the conditioned media of Swan71 human trophoblast cells by ultrafiltration and ultracentrifugation. These were analyzed for density (sucrose density gradient centrifugation), morphology (electron microscopy), size (dynamic light scattering) and protein composition (Ion Trap mass spectrometry and western immunoblotting). Based on density gradient centrifugation, microvesicles from Sw71 cells exhibit a density between 1.134 and 1.173g/ml. Electron microscopy demonstrated that microvesicles from Sw71 cells exhibit the characteristic cup-shaped morphology of exosomes. Dynamic light scattering showed a bell-shaped curve, indicating a homogeneous population with a mean size of 165nm ± 0.5nm. Ion Trap mass spectrometry demonstrated the presence of exosome marker proteins (including CD81, Alix, cytoskeleton related proteins, and Rab family). The MS results were confirmed by western immunoblotting. Based on morphology, density, size and protein composition, we defined the release of exosomes from extravillous trophoblast cells and provide their first extensive characterization. This characterization is essential in furthering our understanding of normal early pregnancy. (hide) EV-METRIC 78% (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 Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Sucrose-DG + UF Protein markers EV: Alix/ CD81/ HSP70/ Beta-actin/ ICAM1/ HSP60 non-EV: Cell organelle protein Proteomics yes EV density (g/ml) 1.13-1.17 Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation 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: time(min) 90 Density gradient Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2 Orientation Top-down Rotor type 90Ti Speed (g) 105000 Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD81/ HSP70/ Beta-actin/ ICAM1/ HSP60 Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins Beta-actin/ ICAM1/ HSP60 Fluorescent NTA Characterization: Particle analysis DLS EM EM-type transmission EM Image type Close-up, Wide-field

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