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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV110095	1/1	Homo sapiens	NAY	(d)(U)C DC Filtration	Hedlund M	2011	11%
Study summary Full title Thermal- and oxidative stress causes enhanced release of NKG2D ligand-bearing immunosuppressive exosomes in leukemia/lymphoma T and B cells All authors Hedlund M, Nagaeva O, Kargl D, Baranov V, Mincheva-Nilsson L Journal PLoS One Abstract Immune evasion from NK surveillance related to inadequate NK-cell function has been suggested as an (show more...)Immune evasion from NK surveillance related to inadequate NK-cell function has been suggested as an explanation of the high incidence of relapse and fatal outcome of many blood malignancies. In this report we have used Jurkat and Raji cell lines as a model for studies of the NKG2D receptor-ligand system in T-and B cell leukemia/lymphoma. Using real-time quantitative RT-PCR and immunoflow cytometry we show that Jurkat and Raji cells constitutively express mRNA and protein for the stress-inducible NKG2D ligands MICA/B and ULBP1 and 2, and up-regulate the expression in a cell-line specific and stress-specific manner. Furthermore, we revealed by electron microscopy, immunoflow cytometry and western blot that these ligands were expressed and secreted on exosomes, nanometer-sized microvesicles of endosomal origin. Acting as a decoy, the NKG2D ligand-bearing exosomes downregulate the in vitro NKG2D receptor-mediated cytotoxicity and thus impair NK-cell function. Interestingly, thermal and oxidative stress enhanced the exosome secretion generating more soluble NKG2D ligands that aggravated the impairment of the cytotoxic response. Taken together, our results might partly explain the clinically observed NK-cell dysfunction in patients suffering from leukemia/lymphoma. The adverse effect of thermal and oxidative stress, enhancing the release of immunosuppressive exosomes, should be considered when cytostatic and hyperthermal anti-cancer therapies are designed. (hide) EV-METRIC 11% (30th 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 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 DC Filtration Protein markers EV: CD63 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 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

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EV-TRACK ID	EV110095
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C DC Filtration
Exp. nr.	1
EV-METRIC %	11