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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV100050	1/1	Homo sapiens	NAY	(d)(U)C Filtration IAF	Park JE	2010	22%
Study summary Full title Hypoxic tumor cell modulates its microenvironment to enhance angiogenic and metastatic potential by secretion of proteins and exosomes All authors Park JE, Tan HS, Datta A, Lai RC, Zhang H, Meng W, Lim SK, Sze SK Journal Mol Cell Proteomics Abstract Under hypoxia, tumor cells produce a secretion that modulates their microenvironment to facilitate t (show more...)Under hypoxia, tumor cells produce a secretion that modulates their microenvironment to facilitate tumor angiogenesis and metastasis. Here, we observed that hypoxic or reoxygenated A431 carcinoma cells exhibited enhanced angiogenic and metastatic potential such as reduced cell-cell and cell-extracellular matrix adhesion, increased invasiveness, and production of a secretion with increased chorioallantoic membrane angiogenic activity. Consistent with these observations, quantitative proteomics revealed that under hypoxia the tumor cells secreted proteins involved in angiogenesis, focal adhesion, extracellular matrix-receptor interaction, and immune cell recruitment. Unexpectedly, the secreted proteins were predominantly cytoplasmic and membrane proteins. Ultracentrifugation at 100,000 x g precipitated 54% of the secreted proteins and enriched for many exosome-associated proteins such as the tetraspanins and Alix and also proteins with the potential to facilitate angiogenesis and metastasis. Two tetraspanins, CD9 and CD81, co-immunoprecipitated. Together, these data suggested that tumor cells secrete proteins and exosomes with the potential to modulate their microenvironment and facilitate angiogenesis and metastasis. (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 IAF Adj. k-factor 255.8 (pelleting) Protein markers EV: Alix/ CD81/ CD9 non-EV: Proteomics yes 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 Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW41 Pelleting: adjusted k-factor 255.8 Filtration steps 0.22µm or 0.2µm Immunoaffinity capture Selected surface protein(s) CD81 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD81/ CD9

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EV-TRACK ID	EV100050
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C Filtration IAF
Exp. nr.	1
EV-METRIC %	22