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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200190	1/1	Homo sapiens	HEK293	(d)(U)C DG	Pečan, Peter	2020	67%
Study summary Full title Calcium Ionophore-Induced Extracellular Vesicles Mediate Cytoprotection against Simulated Ischemia/Reperfusion Injury in Cardiomyocyte-Derived Cell Lines by Inducing Heme Oxygenase 1 All authors Peter Pečan, Szabolcs Hambalkó, Van Thai Ha, Csilla T Nagy, Csilla Pelyhe, Duško Lainšček, Bence Kenyeres, Gábor B Brenner, Anikó Görbe, Ágnes Kittel, Monika Barteková, Péter Ferdinandy, Mateja Manček-Keber, Zoltán Giricz Journal Int J Mol Sci Abstract Cardioprotection against ischemia/reperfusion injury is still an unmet clinical need. The transient (show more...)Cardioprotection against ischemia/reperfusion injury is still an unmet clinical need. The transient activation of Toll-like receptors (TLRs) has been implicated in cardioprotection, which may be achieved by treatment with blood-derived extracellular vesicles (EVs). However, since the isolation of EVs from blood takes considerable effort, the aim of our study was to establish a cellular model from which cardioprotective EVs can be isolated in a well-reproducible manner. EV release was induced in HEK293 cells with calcium ionophore A23187. EVs were characterized and cytoprotection was assessed in H9c2 and AC16 cell lines. Cardioprotection afforded by EVs and its mechanism were investigated after 16 h simulated ischemia and 2 h reperfusion. The induction of HEK293 cells by calcium ionophore resulted in the release of heterogenous populations of EVs. In H9c2 and AC16 cells, stressEVs induced the downstream signaling of TLR4 and heme oxygenase 1 (HO-1) expression in H9c2 cells. StressEVs decreased necrosis due to simulated ischemia/reperfusion injury in H9c2 and AC16 cells, which was independent of TLR4 induction, but not that of HO-1. Calcium ionophore-induced EVs exert cytoprotection by inducing HO-1 in a TLR4-independent manner. (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. 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 calcium ionophore A23187 Focus vesicles extracellular vesicle 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 (Differential) (ultra)centrifugation Density gradient Protein markers EV: calnexin/ TSG101/ annexin V/ Alix/ CD81 non-EV: histone H3 Proteomics no EV density (g/ml) 1.088-1.117 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type Type 50 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 60 Wash: Rotor Type Type 50 Ti Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 0.250 Orientation Top-down Rotor type Type 50 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing None Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins annexin V/ TSG101/ Alix/ CD81 Detected contaminants histone H3/ calnexin Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) <100-1000 NTA Report type Mean Reported size (nm) 250 EM EM-type Transmission-EM Image type Close-up

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EV-TRACK ID	EV200190
species	Homo sapiens
sample type	Cell culture
cell type	HEK293
condition	calcium ionophore A23187
separation protocol	dUC Density gradient
Exp. nr.	1
EV-METRIC %	67