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Results list Most used separation protocols Top EV-enriched proteins

Details	EV-TRACK ID	Experiment nr.	Species	Sample type	separation protocol	First author	Year	EV-METRIC
	EV180053	1/1	Homo sapiens	Cell culture supernatant	(d)(U)C Filtration	Rocha, Sara	2018	75%
Study summary Full title 3D Cellular Architecture Affects MicroRNA and Protein Cargo of Extracellular Vesicles All authors Sara Rocha, Joana Carvalho, Patrícia Oliveira, Maren Voglstaetter, Domitille Schvartz, Andreas R. Thomsen, Nadia Walter, Richa Khanduri, Jean‐Charles Sanchez, Andreas Keller, Carla Oliveira, Irina Nazarenko Journal Advanced Science Abstract The success of malignant tumors is conditioned by the intercellular communication between tumor cell (show more...)The success of malignant tumors is conditioned by the intercellular communication between tumor cells and their microenvironment, with extracellular vesicles (EVs) acting as main mediators. While the value of 3D conditions to study tumor cells is well established, the impact of cellular architecture on EV content and function is not investigated yet. Here, a recently developed 3D cell culture microwell array is adapted for EV production and a comprehensive comparative analysis of biochemical features, RNA and proteomic profiles of EVs secreted by 2D vs 3D cultures of gastric cancer cells, is performed. 3D cultures are significantly more efficient in producing EVs than 2D cultures. Global upregulation of microRNAs and downregulation of proteins in 3D are observed, indicating their dynamic coregulation in response to cellular architecture, with the ADP‐ribosylation factor 6 signaling pathway significantly downregulated in 3D EVs. The data strengthen the biological relevance of cellular architecture for production and cargo of EVs. (hide) EV-METRIC 75% (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 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 Cell Name MKN74, MKN45 Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: integrin-alpha6/ catenin-beta1/ integrin-alpha2/ integrin-alpha3/ tubulin/ Flotillin-1/ E-cadherin/ GAPDH/ CD81/ integrin-beta1/ catenin-delta1/ CD9 non-EV: CytochromeC Proteomics yes Show all info Study aim New methodological development, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells MKN74, MKN45 EV-harvesting Medium Serum free medium Cell viability 95 Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Concentration 0.2 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins integrin-alpha2, integrin-alpha3, integrin-alpha6, integrin-beta1, E-cadherin, catenin-beta1, catenin-delta1, GAPDH, tubulin Proteomics Proteomics database Yes Characterization: RNA analysis Proteinase treatment Moment of Proteinase treatment After Proteinase type Proteinase K Proteinase concentration 5 RNAse treatment Moment of RNAse treatment After RNAse type RNase A RNAse concentration 1 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 110 EV concentration Yes Particle yield 1.50E+09 particles/million cells Particle analysis: flow cytometry Flow cytometer type Amnis ImageStream Hardware adjustment Calibration bead size 200 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 110

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