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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV130042	1/1	Mus musculus	NAY	(d)(U)C DG Filtration	Kotzerke K	2013	56%
Study summary Full title Immunostimulatory activity of murine keratinocyte-derived exosomes All authors Kotzerke K, Mempel M, Aung T, Wulf GG, Urlaub H, Wenzel D, Schön MP, Braun A Journal Exp Dermatol Abstract It has long been known that keratinocytes influence cutaneous immunity through secretion of soluble (show more...)It has long been known that keratinocytes influence cutaneous immunity through secretion of soluble factors. Exosomes, small membrane vesicles of endocytotic origin, have been implicated in intercellular communication processes such as the transfer of tumor cell antigens and the activation of recipient dendritic cells (DC). However, little is known about immunomodulatory functions of keratinocyte-derived exosomes. To address this question, we analysed exosome secretion of the murine keratinocyte cell line MPEK under steady state as well as inflammatory conditions (+/- IFN?). These exosomes were readily taken up by bone marrow-derived DC (BMDC) in vitro resulting in a matured phenotype, as evidenced by increased CD40 expression as well as by the production of large amounts of IL-6, IL-10 and IL-12. When the transfer of antigen-specific information through exosomes was investigated, it was found that keratinocytes took up antigen (ovalbumin) and transferred it to their exosomes. However, these antigen-harbouring exosomes failed to induce antigen-specific T cell responses via BMDC. Together, this novel biological function suggests that keratinocytes are able to direct unspecific immune processes but do not elicit specific immune responses. (hide) EV-METRIC 56% (90th 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 DG Filtration Adj. k-factor 256 (pelleting) Protein markers EV: Alix/ Flotillin non-EV: Cell organelle protein Proteomics yes EV density (g/ml) 1.100 Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps 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) 150 Pelleting: rotor type SW32 Pelleting: adjusted k-factor 256.0 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2.25 Orientation Top-down Rotor type SW32 Speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ Flotillin Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins Flotillin Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

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EV-TRACK ID	EV130042
species	Mus musculus
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C DG Filtration
Exp. nr.	1
EV-METRIC %	56