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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200007	1/1	Homo sapiens	breast milk	DG (d)(U)C SEC	Zonneveld, Marijke	2021	75%
Study summary Full title Human milk extracellular vesicles target nodes in interconnected signalling pathways that enhance oral epithelial barrier function and dampen immune responses All authors Marijke I Zonneveld, Martijn J C van Herwijnen, Marcela M Fernandez-Gutierrez, Alberta Giovanazzi, Anne Marit de Groot, Marije Kleinjan, Toni M M van Capel, Alice J A M Sijts, Leonie S Taams, Johan Garssen, Esther C de Jong, Michiel Kleerebezem, Esther N M Nolte-'t Hoen, Frank A Redegeld, Marca H M Wauben Journal J Extracell Vesicles Abstract Maternal milk is nature's first functional food. It plays a crucial role in the development of the i (show more...)Maternal milk is nature's first functional food. It plays a crucial role in the development of the infant's gastrointestinal (GI) tract and the immune system. Extracellular vesicles (EVs) are a heterogeneous population of lipid bilayer enclosed vesicles released by cells for intercellular communication and are a component of milk. Recently, we discovered that human milk EVs contain a unique proteome compared to other milk components. Here, we show that physiological concentrations of milk EVs support epithelial barrier function by increasing cell migration via the p38 MAPK pathway. Additionally, milk EVs inhibit agonist-induced activation of endosomal Toll like receptors TLR3 and TLR9. Furthermore, milk EVs directly inhibit activation of CD4+ T cells by temporarily suppressing T cell activation without inducing tolerance. We show that milk EV proteins target key hotspots of signalling networks that can modulate cellular processes in various cell types of the GI tract. (hide) EV-METRIC 75% (87th 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 breast milk 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. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C SEC Protein markers EV: CD63/ Flotillin1/ CD9/ HSP70 non-EV: Lactoferrin Proteomics no EV density (g/ml) 1.06-1.19 Show all info Study aim Function Sample Species Homo sapiens Sample Type breast milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 10% Highest density fraction 60% Total gradient volume, incl. sample (mL) 12.5 Sample volume (mL) 6.5 Orientation Top-down Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 900 Fraction volume (mL) 0.5 Fraction processing Size-exclusion chromatography Size-exclusion chromatography Total column volume (mL) 15 Sample volume/column (mL) 2.5 Resin type Sephadex G-100 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ CD9/ CD63/ HSP70 Detected contaminants Lactoferrin Characterization: Lipid analysis No

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EV-TRACK ID	EV200007
species	Homo sapiens
sample type	breast milk
condition	Control condition
separation protocol	DG (d)(U)C SEC
Exp. nr.	1
EV-METRIC %	75