EV-TRACK

Search > Results

You searched for: EV100011 (EV-TRACK ID)

Showing 1 - 2 of 2

Results list Study Tree

Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV100011	2/2	Homo sapiens	NAY	(d)(U)C DG Filtration IAF	Barreto A	2010	67%
Study summary Full title Membrane vesicles released by intestinal epithelial cells infected with rotavirus inhibit T-cell function All authors Barreto A, Rodríguez LS, Rojas OL, Wolf M, Greenberg HB, Franco MA, Angel J Journal Viral Immunol Abstract Rotavirus (RV) predominantly replicates in intestinal epithelial cells (IEC), and danger signals rel (show more...)Rotavirus (RV) predominantly replicates in intestinal epithelial cells (IEC), and danger signals released by these cells may modulate viral immunity. We have recently shown that human model IEC (Caco-2 cells) infected with rhesus-RV release a non-inflammatory group of immunomodulators that includes heat shock proteins (HSPs) and TGF-?1. Here we show that both proteins are released in part in association with membrane vesicles (MV) obtained from filtrated Caco-2 supernatants concentrated by ultracentrifugation. These MV express markers of exosomes (CD63 and others), but not of the endoplasmic reticulum (ER) or nuclei. Larger quantities of proteins associated with MV were released by RV-infected cells than by non-infected cells. VP6 co-immunoprecipitated with CD63 present in these MV, and VP6 co-localized with CD63 in RV-infected cells, suggesting that this viral protein is associated with the MV, and that this association occurs intracellularly. CD63 present in MV preparations from stool samples from 36 children with gastroenteritis due or not due to RV were analyzed. VP6 co-immunoprecipitated with CD63 in 3/8 stool samples from RV-infected children, suggesting that these MV are released by RV-infected cells in vivo. Moreover, fractions that contained MV from RV-infected cells induced death and inhibited proliferation of CD4(+) T cells to a greater extent than fractions from non-infected cells. These effects were in part due to TGF-?, because they were reversed by treatment of the T cells with the TGF-?-receptor inhibitor ALK5i. MV from RV-infected and non-infected cells were heterogeneous, with morphologies and typical flotation densities described for exosomes (between 1.10 and 1.18 g/mL), and denser vesicles (>1.24 g/mL). Both types of MV from RV-infected cells were more efficient at inhibiting T-cell function than were those from non-infected cells. We propose that RV infection of IEC releases MV that modulate viral immunity. (hide) EV-METRIC 67% (94th 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 Membrane(-derived) vesicles 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 IAF Adj. k-factor 156.9 (pelleting) Protein markers EV: AChE/ CD63/ MFGE8/ HSC70/ HSP70 non-EV: Cell organelle protein Proteomics no EV density (g/ml) 1.1-1.18;1.24-1.3 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 156.9 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2 Orientation Bottom-up Speed (g) 100000 Filtration steps 0.22µm or 0.2µm Immunoaffinity capture Selected surface protein(s) CD63 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ HSP70/ AChE/ HSC70/ MFGE8 Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins AChE/ HSC70/ MFGE8 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV100011	1/2	Homo sapiens	Stool	(d)(U)C Filtration IAF	Barreto A	2010	22%
Study summary Full title Membrane vesicles released by intestinal epithelial cells infected with rotavirus inhibit T-cell function All authors Barreto A, Rodríguez LS, Rojas OL, Wolf M, Greenberg HB, Franco MA, Angel J Journal Viral Immunol Abstract Rotavirus (RV) predominantly replicates in intestinal epithelial cells (IEC), and danger signals rel (show more...)Rotavirus (RV) predominantly replicates in intestinal epithelial cells (IEC), and danger signals released by these cells may modulate viral immunity. We have recently shown that human model IEC (Caco-2 cells) infected with rhesus-RV release a non-inflammatory group of immunomodulators that includes heat shock proteins (HSPs) and TGF-?1. Here we show that both proteins are released in part in association with membrane vesicles (MV) obtained from filtrated Caco-2 supernatants concentrated by ultracentrifugation. These MV express markers of exosomes (CD63 and others), but not of the endoplasmic reticulum (ER) or nuclei. Larger quantities of proteins associated with MV were released by RV-infected cells than by non-infected cells. VP6 co-immunoprecipitated with CD63 present in these MV, and VP6 co-localized with CD63 in RV-infected cells, suggesting that this viral protein is associated with the MV, and that this association occurs intracellularly. CD63 present in MV preparations from stool samples from 36 children with gastroenteritis due or not due to RV were analyzed. VP6 co-immunoprecipitated with CD63 in 3/8 stool samples from RV-infected children, suggesting that these MV are released by RV-infected cells in vivo. Moreover, fractions that contained MV from RV-infected cells induced death and inhibited proliferation of CD4(+) T cells to a greater extent than fractions from non-infected cells. These effects were in part due to TGF-?, because they were reversed by treatment of the T cells with the TGF-?-receptor inhibitor ALK5i. MV from RV-infected and non-infected cells were heterogeneous, with morphologies and typical flotation densities described for exosomes (between 1.10 and 1.18 g/mL), and denser vesicles (>1.24 g/mL). Both types of MV from RV-infected cells were more efficient at inhibiting T-cell function than were those from non-infected cells. We propose that RV infection of IEC releases MV that modulate viral immunity. (hide) EV-METRIC 22% (50th 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 Stool Sample origin NAY Focus vesicles Membrane(-derived) vesicles 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 IAF Adj. k-factor 156.9 (pelleting) Protein markers EV: CD63 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Stool 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 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 156.9 Filtration steps 0.22µm or 0.2µm Immunoaffinity capture Selected surface protein(s) CD63 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63 Characterization: Particle analysis None

				1 - 2 of 2

EV-TRACK ID	EV100011
species	Homo sapiens
sample type	Cell culture	Stool
cell type	NAY	NA
medium	serum free
condition	NAY	NAY
separation protocol	(d)(U)C DG Filtration IAF	(d)(U)C Filtration IAF
Exp. nr.	2	1
EV-METRIC %	67	22