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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230964	1/1	Homo sapiens	human milk	ExoQuick Filtration (d)(U)C	Bickmore DC	2020	50%
Study summary Full title Characterization of Extracellular Vesicles Isolated From Human Milk Using a Precipitation-Based Method. All authors Bickmore DC, Miklavcic JJ Journal Front Nutr Abstract Extracellular vesicles (EV) function in intercellular communication, and those in human milk may con (show more...)Extracellular vesicles (EV) function in intercellular communication, and those in human milk may confer immunologic benefits to infants. Methods of EV isolation such as ultracentrifugation (UC) may not be feasible for the study of EVs in human milk due to the need for large sample volume. A technique to isolate EVs from a small volume of human milk using a precipitation reagent is described herein. Electron microscopy, nanoparticle tracking analysis, and semi-quantitative antibody array were conducted to confirm isolation of human milk EVs. Count, size, protein content, and fatty acid quantification of EVs were determined. This isolation technique yielded 8.9 x 10 (± 1.1 × 10) EV particles/mL of human milk. The present method meets the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines. An established EV isolation method suitable for a low volume of human milk will facilitate further research in this growing area. (hide) EV-METRIC 50% (25th 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 human 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 ExoQuick Filtration (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD63/ CD81/ Flotillin1/ TSG101/ anxa5/ EpCAM/ ICAM1 non-EV: GM130 Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type human 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 Filtration steps Between 0.22 and 0.45 µm Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Detected EV-associated proteins Alix/ CD63/ CD81/ Flotillin1/ TSG101/ anxa5/ EpCAM/ ICAM1 Not detected contaminants GM130 Characterization: Lipid analysis Yes Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 200-278 NTA Report type Modus Reported size (nm) 150 EV concentration Yes Particle yield particles per milliliter of starting sample: 8.9E9 EM EM-type Scanning-EM Image type Wide-field Report size (nm) 50-350

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EV-TRACK ID	EV230964
species	Homo sapiens
sample type	human milk
condition	Control condition
separation protocol	ExoQuick/ Filtration/ dUC
Exp. nr.	1
EV-METRIC %	50