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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV100017	1/2	Homo sapiens	NAY	(d)(U)C DG IAF	Esser J	2010	44%
Study summary Full title Exosomes from human macrophages and dendritic cells contain enzymes for leukotriene biosynthesis and promote granulocyte migration All authors Esser J, Gehrmann U, D'Alexandri FL, Hidalgo-Estévez AM, Wheelock CE, Scheynius A, Gabrielsson S, Rådmark O Journal J Allergy Clin Immunol Abstract BACKGROUND: Leukotrienes (LTs) are potent proinflammatory lipid mediators with key roles in the path (show more...)BACKGROUND: Leukotrienes (LTs) are potent proinflammatory lipid mediators with key roles in the pathogenesis of asthma and inflammation. Recently, nanovesicles (exosomes), released from macrophages and dendritic cells (DCs), have become increasingly appreciated as messengers in immunity. OBJECTIVE: We investigated whether exosomes from human macrophages, DCs, and plasma contain enzymes for LT biosynthesis and studied potential roles for exosomes in transcellular LT metabolism and granulocyte chemotaxis. METHODS: The presence of LT pathway enzymes and LT biosynthesis in exosomes and cells was analyzed by Western blot, immunoelectron microscopy, and enzyme activity assays. Surface marker expression was evaluated by flow cytometry, and granulocyte migration was assessed in a multiwell chemotaxis system. RESULTS: Exosomes from macrophages and DCs contain functional enzymes for LT biosynthesis. After incubation of intact cells with the LT biosynthesis intermediate LTA(4), LTB(4) was the major product of macrophages, whereas DCs primarily formed LTC(4). However, in exosomes from both cell types, LTC(4) was the predominant LTA(4) metabolite. Exosomal LTC(4) formation (per milligram protein) exceeded that of cells. In macrophages and DCs, TGF-?1 upregulated LTA(4) hydrolase along with increased LTB(4) formation also in the exosomes. Moreover, TGF-?1 modified the expression of surface marker proteins on cells and exosomes and reduced the exosome yield from macrophages. On Ca(2+)-ionophore and arachidonic acid stimulation, exosomes produced chemotactic eicosanoids and induced granulocyte migration. Interestingly, active LTA(4) hydrolase and LTC(4) synthase were present also in exosomes from human plasma. CONCLUSION: Our findings indicate that exosomes can contribute to inflammation by participation in LT biosynthesis and granulocyte recruitment. (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 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 IAF Protein markers EV: CD81/ CD63/ Beta-actin/ MHC2 non-EV: Proteomics no EV density (g/ml) 1.13-1.19 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 Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2 Orientation Top-down Speed (g) 100000 Immunoaffinity capture Selected surface protein(s) MHC2 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ MHC2/ Beta-actin ELISA Antibody details provided? No Detected EV-associated proteins MHC2/ Beta-actin Characterization: Particle analysis EM EM-type immune EM EM protein LTC4S Image type Close-up

	EV100017	2/2	Homo sapiens	NAY	(d)(U)C Filtration	Esser J	2010	22%
Study summary Full title Exosomes from human macrophages and dendritic cells contain enzymes for leukotriene biosynthesis and promote granulocyte migration All authors Esser J, Gehrmann U, D'Alexandri FL, Hidalgo-Estévez AM, Wheelock CE, Scheynius A, Gabrielsson S, Rådmark O Journal J Allergy Clin Immunol Abstract BACKGROUND: Leukotrienes (LTs) are potent proinflammatory lipid mediators with key roles in the path (show more...)BACKGROUND: Leukotrienes (LTs) are potent proinflammatory lipid mediators with key roles in the pathogenesis of asthma and inflammation. Recently, nanovesicles (exosomes), released from macrophages and dendritic cells (DCs), have become increasingly appreciated as messengers in immunity. OBJECTIVE: We investigated whether exosomes from human macrophages, DCs, and plasma contain enzymes for LT biosynthesis and studied potential roles for exosomes in transcellular LT metabolism and granulocyte chemotaxis. METHODS: The presence of LT pathway enzymes and LT biosynthesis in exosomes and cells was analyzed by Western blot, immunoelectron microscopy, and enzyme activity assays. Surface marker expression was evaluated by flow cytometry, and granulocyte migration was assessed in a multiwell chemotaxis system. RESULTS: Exosomes from macrophages and DCs contain functional enzymes for LT biosynthesis. After incubation of intact cells with the LT biosynthesis intermediate LTA(4), LTB(4) was the major product of macrophages, whereas DCs primarily formed LTC(4). However, in exosomes from both cell types, LTC(4) was the predominant LTA(4) metabolite. Exosomal LTC(4) formation (per milligram protein) exceeded that of cells. In macrophages and DCs, TGF-?1 upregulated LTA(4) hydrolase along with increased LTB(4) formation also in the exosomes. Moreover, TGF-?1 modified the expression of surface marker proteins on cells and exosomes and reduced the exosome yield from macrophages. On Ca(2+)-ionophore and arachidonic acid stimulation, exosomes produced chemotactic eicosanoids and induced granulocyte migration. Interestingly, active LTA(4) hydrolase and LTC(4) synthase were present also in exosomes from human plasma. CONCLUSION: Our findings indicate that exosomes can contribute to inflammation by participation in LT biosynthesis and granulocyte recruitment. (hide) EV-METRIC 22% (59th 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 Filtration Protein markers EV: Beta-actin 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 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 Beta-actin ELISA Antibody details provided? No Detected EV-associated proteins Beta-actin Characterization: Particle analysis None

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EV-TRACK ID	EV100017
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C DG IAF	(d)(U)C Filtration
Exp. nr.	1	2
EV-METRIC %	44	22