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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV110115	1/1	Mus musculus	NAY	(d)(U)C Filtration UF	Sheng H	2011	11%
Study summary Full title Insulinoma-released exosomes or microparticles are immunostimulatory and can activate autoreactive T cells spontaneously developed in nonobese diabetic mice All authors Sheng H, Hassanali S, Nugent C, Wen L, Hamilton-Williams E, Dias P, Dai YD Journal J Immunol Abstract Exosomes (EXO) are secreted intracellular microparticles that can trigger inflammation and induce Ag (show more...)Exosomes (EXO) are secreted intracellular microparticles that can trigger inflammation and induce Ag-specific immune responses. To test possible roles of EXO in autoimmunity, we isolated small microparticles, mainly EXO, from mouse insulinoma and examined their activities to stimulate the autoimmune responses in NOD mice, a model for human type 1 diabetes. We demonstrate that the EXO contains strong innate stimuli and expresses candidate diabetes autoantigens. They can induce secretion of inflammatory cytokines through a MyD88-dependent pathway, and activate purified APC and result in T cell proliferation. To address whether EXO or the secreted microparticles are possible autoimmune targets causing islet-specific inflammation, we monitored the T cell responses spontaneously developed in prediabetic NOD mice for their reactivity to the EXO, and compared this reactivity between diabetes-susceptible and -resistant congenic mouse strains. We found that older NOD females, which have advanced islet destruction, accumulated more EXO-reactive, IFN-?-producing lymphocytes than younger females or age-matched males, and that pancreatic lymph nodes from the prediabetic NOD, but not from the resistant mice, were also enriched with EXO-reactive Th1 cells. In vivo, immunization with the EXO accelerates insulitis development in nonobese diabetes-resistant mice. Thus, EXO or small microparticles can be recognized by the diabetes-associated autoreactive T cells, supporting that EXO might be a possible autoimmune target and/or insulitis trigger in NOD or congenic mouse strains. (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 / microparticles 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: GAD65/ HSC70 non-EV: Proteomics yes Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted 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) 90 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins GAD65/ HSC70 ELISA Antibody details provided? No Detected EV-associated proteins GAD65/ HSC70 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

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EV-TRACK ID	EV110115
species	Mus musculus
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C Filtration UF
Exp. nr.	1
EV-METRIC %	11