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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV110084	1/1	Homo sapiens	NAY	(d)(U)C Filtration UF	Atay S	2011	0%
Study summary Full title Human trophoblast-derived exosomal fibronectin induces pro-inflammatory IL-1beta production by macrophages All authors Atay S, Gercel-Taylor C, Taylor DD Journal Am J Reprod Immunol Abstract PROBLEM Our previous studies demonstrated that trophoblast-derived exosomes induced synthesis and re (show more...)PROBLEM Our previous studies demonstrated that trophoblast-derived exosomes induced synthesis and release of pro-inflammatory cytokines, including interleukin-1? (IL-1?) by macrophages. The objective of this study was to characterize the mechanism and receptors associated with this induction. METHOD OF STUDY Exosomes were isolated from Sw71 trophoblast-conditioned media by ultrafiltration and ultracentrifugation. Using macrophages isolated from normal donors, cytochalasin D was used to block exosome uptake. Induction of IL-1? mRNA was investigated by qRT-PCR, pro-IL-1? protein by western immunoblotting, and mature IL-1? release by ELISA. RGD peptides were used to block fibronectin binding by macrophage ?5?1 integrin. RESULTS Uptake of exosomes by macrophages was completely blocked by pre-treatment with cytochalasin D. Although induction of some cytokines (such as C4A and CCL11) requires uptake, induction of IL-1? occurred without exosome internalization. Cytochalasin D treatment did not inhibit exosome-mediated induction of IL-1? mRNA, production of the pro-protein, or release of mature IL-1?. Blocking of fibronectin binding using RGD peptides demonstrated the abrogation of exosome-mediated IL-1? production. CONCLUSION Although trophoblast-derived exosomes have been demonstrated to induce IL-1?, this is the first demonstration of IL-1? induction by exosome-associated fibronectin. Based on this pro-inflammatory role of exosome-associated fibronectin, it may represent an important general immunoregulatory mechanism. (hide) EV-METRIC 0% (median: 14% 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 NAY Focus vesicles exosomes 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 (d)(U)C Filtration UF Protein markers EV: non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 90 Filtration steps 0.22µm or 0.2µm

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EV-TRACK ID	EV110084
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C Filtration UF
Exp. nr.	1
EV-METRIC %	0