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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV120036	1/1	Homo sapiens	NAY	(d)(U)C	Palazzolo G	2012	44%
Study summary Full title Proteomic analysis of exosome-like vesicles derived from breast cancer cells All authors Palazzolo G, Albanese NN, DI Cara G, Gygax D, Vittorelli ML, Pucci-Minafra I Journal Anticancer Res Abstract BACKGROUND/AIM: The phenomenon of membrane vesicle-release by neoplastic cells is a growing field of (show more...)BACKGROUND/AIM: The phenomenon of membrane vesicle-release by neoplastic cells is a growing field of interest in cancer research, due to their potential role in carrying a large array of tumor antigens when secreted into the extracellular medium. In particular, experimental evidence show that at least some of the tumor markers detected in the blood circulation of mammary carcinoma patients are carried by membrane-bound vesicles. Thus, biomarker research in breast cancer can gain great benefits from vesicle characterization. MATERIALS AND METHODS: Conditioned medium was collected from serum starved MDA-MB-231 sub-confluent cell cultures and exosome-like vesicles (ELVs) were isolated by ultracentrifugation. Ultrastructural analysis of ELVs was performed by transmission electron microscopy (TEM) and the purity of fraction was confirmed by western blotting assays. Proteomic profile of ELVs was carried out by 2 D-PAGE and protein identification performed by MALDI-ToF Mass Spectrometry. RESULTS: On the basis of ultrastructural and immunological characterization, the isolated vesicles have been classified as exosome-like vesicles (ELVs). The proteomic investigation showed a distinctive protein profile of the ELVs, in comparison to the whole cell lisates (WCL) proteome, which could be instrumental for cancer progression. The proteins were clustered into functional categories, according to the current bioinformatics resources and a Venn diagram was constructed based on these clusters. CONCLUSION: It is reasonable to assume that vesicle production allows neoplastic cells to exert different effects, according to the possible acceptor targets. For instance, vesicles could potentiate the malignant properties of adjacent neoplastic cells or activate non-tumoral cells. Moreover, vesicles could convey signals to immune cells and surrounding stroma cells. The present study may significantly contribute to the knowledge of the vesiculation phenomenon, which is a critical device for trans cellular communication in cancer. (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 Exosome-like 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 Adj. k-factor 159.6 (pelleting) Protein markers EV: LAMP1/ HSC70 non-EV: Cell organelle protein Proteomics yes TEM measurements 40-80 Show all info Study aim Omics Sample Species Homo sapiens 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) 90 Pelleting: rotor type 60Ti Pelleting: adjusted k-factor 159.6 Characterization: Protein analysis Western Blot Detected EV-associated proteins HSC70/ LAMP1 Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins HSC70/ LAMP1 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field Report size (nm) 40-80

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EV-TRACK ID	EV120036
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C
Exp. nr.	1
EV-METRIC %	44