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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140047	2/2	Homo sapiens	NAY	(d)(U)C Filtration	Gonzalez E	2014	44%
Study summary Full title Human mammospheres secrete hormone-regulated active extracellular vesicles All authors Gonzalez E, Piva M, Rodriguez-Suarez E, Gil D, Royo F, Elortza F, Falcon-Perez JM, Vivanco Md Journal PLoS One Abstract Breast cancer is a leading cause of cancer-associated death worldwide. One of the most important pro (show more...)Breast cancer is a leading cause of cancer-associated death worldwide. One of the most important prognostic factors for survival is the early detection of the disease. Recent studies indicate that extracellular vesicles may provide diagnostic information for cancer management. We demonstrate the secretion of extracellular vesicles by primary breast epithelial cells enriched for stem/progenitor cells cultured as mammospheres, in non-adherent conditions. Using a proteomic approach we identified proteins contained in these vesicles whose expression is affected by hormonal changes in the cellular environment. In addition, we showed that these vesicles are capable of promoting changes in expression levels of genes involved in epithelial-mesenchymal transition and stem cell markers. Our findings suggest that secreted extracellular vesicles could represent potential diagnostic and/or prognostic markers for breast cancer and support a role for extracellular vesicles in cancer progression. (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 extracellular 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 Protein markers EV: CD63/ CD133/ MFGE8/ CD81/ Flotilin1/ Annexin2/ CD13 non-EV: Cell organelle protein 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 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ Flotilin1/ MFGE8/ CD13/ CD133/ Annexin2 Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins MFGE8/ CD13/ CD133/ Annexin2 Characterization: Particle analysis NTA EM EM-type cryo EM Image type Close-up

	EV140047	1/2	Homo sapiens	NAY	(d)(U)C Filtration	Gonzalez E	2014	33%
Study summary Full title Human mammospheres secrete hormone-regulated active extracellular vesicles All authors Gonzalez E, Piva M, Rodriguez-Suarez E, Gil D, Royo F, Elortza F, Falcon-Perez JM, Vivanco Md Journal PLoS One Abstract Breast cancer is a leading cause of cancer-associated death worldwide. One of the most important pro (show more...)Breast cancer is a leading cause of cancer-associated death worldwide. One of the most important prognostic factors for survival is the early detection of the disease. Recent studies indicate that extracellular vesicles may provide diagnostic information for cancer management. We demonstrate the secretion of extracellular vesicles by primary breast epithelial cells enriched for stem/progenitor cells cultured as mammospheres, in non-adherent conditions. Using a proteomic approach we identified proteins contained in these vesicles whose expression is affected by hormonal changes in the cellular environment. In addition, we showed that these vesicles are capable of promoting changes in expression levels of genes involved in epithelial-mesenchymal transition and stem cell markers. Our findings suggest that secreted extracellular vesicles could represent potential diagnostic and/or prognostic markers for breast cancer and support a role for extracellular vesicles in cancer progression. (hide) EV-METRIC 33% (75th 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 extracellular 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 Protein markers EV: Flotilin1/ CD63/ CD133/ CD13 non-EV: Proteomics no 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 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotilin1/ CD133/ CD13 ELISA Antibody details provided? No Detected EV-associated proteins CD133/ CD13 Characterization: Particle analysis NTA EM EM-type cryo EM Image type Wide-field

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EV-TRACK ID	EV140047
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C Filtration	(d)(U)C Filtration
Exp. nr.	2	1
EV-METRIC %	44	33