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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV100007	1/1	Mus musculus	NAY	(d)(U)C UF	Ramachandra L	2010	44%
Study summary Full title Mycobacterium tuberculosis synergizes with ATP to induce release of microvesicles and exosomes containing major histocompatibility complex class II molecules capable of antigen presentation All authors Ramachandra L, Qu Y, Wang Y, Lewis CJ, Cobb BA, Takatsu K, Boom WH, Dubyak GR, Harding CV Journal Infect Immun Abstract Major histocompatibility complex class II (MHC-II) molecules are released by murine macrophages upon (show more...)Major histocompatibility complex class II (MHC-II) molecules are released by murine macrophages upon lipopolysaccharide (LPS) stimulation and ATP signaling through the P2X7 receptor. These studies show that infection of macrophages with Mycobacterium tuberculosis or M. bovis strain BCG enhances MHC-II release in synergy with ATP. Shed MHC-II was contained in two distinct organelles, exosomes and plasma membrane-derived microvesicles, which were both able to present exogenous antigenic peptide to T hybridoma cells. Furthermore, microvesicles from mycobacterium-infected macrophages were able to directly present M. tuberculosis antigen (Ag) 85B(241-256)-I-A(b) complexes that were generated by the processing of M. tuberculosis Ag 85B in infected cells to both M. tuberculosis-specific T hybridoma cells and naïve P25 M. tuberculosis T-cell receptor (TCR)-transgenic T cells. In the presence of prefixed macrophages, exosomes from mycobacterium-infected macrophages provided weak stimulation to M. tuberculosis-specific T hybridoma cells but not naïve P25 T cells. Thus, infection with M. tuberculosis primes macrophages for the increased release of exosomes and microvesicles bearing M. tuberculosis peptide-MHC-II complexes that may generate antimicrobial T-cell responses. (hide) EV-METRIC 44% (84th 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 / microvesicles 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 UF Adj. k-factor 157.1 (pelleting) Protein markers EV: MHC2/ Rab7/ ICAM1/ CD86 non-EV: Cell organelle protein Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant 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) 900 Pelleting: rotor type 50.2Ti Pelleting: adjusted k-factor 157.1 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins MHC2/ CD86/ ICAM1/ Rab7 Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins MHC2/ CD86/ ICAM1/ Rab7 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

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EV-TRACK ID	EV100007
species	Mus musculus
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C UF
Exp. nr.	1
EV-METRIC %	44