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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210145	1/1	Homo sapiens	human milk	(d)(U)C	Wang, John	2022	56%
Study summary Full title Quantitation of Exosomes and Their MicroRNA Cargos in Frozen Human Milk All authors Haichuan Wang, Di Wu, Sonal Sukreet, Anthony Delaney, Mandy Belfort, Janos Zempleni Journal Journal of pediatric gastroenterology and nutrition Abstract We assessed feasibility of analyzing exosomes and microRNA cargos in frozen human milk as a prerequi (show more...)We assessed feasibility of analyzing exosomes and microRNA cargos in frozen human milk as a prerequisite for epidemiological studies of milk exosomes. We collected milk from 5 mother-preterm infant dyads at 3 time points during postnatal hospital care for storage at −80 °C. We purified exosomes by ultracentrifugation, probed marker proteins using immunoblots, assessed size and counts with a nanoparticle tracker, and quantified 3 microRNAs with quantitative PCR. Positive exosome marker proteins were detectable; β-casein was the only detectable contaminant. Exosome count and size trended to decrease from early to late samples (count, 2.3 × 109 ± 3.8 × 109 to 5.6 × 108 ± 9.7 × 108 exosomes/mL; size, 117 ± 25 to 92 ± 16 nm). Two microRNAs were detectable in early samples only; cycle threshold values equaled 28.7 ± 0.7 for miR-30d-5p and miR-125a-5p; miR-423-5p was not detectable. We conclude that the analysis of exosomes and quantification of microRNAs is feasible in human milk previously stored at −80 °C. (hide) EV-METRIC 56% (50th 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 exosome 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 Protein markers EV: Alix/ CD63/ CD9 non-EV: beta casein/ alfa tubulin/ intergrin beta Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type human milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Thermo F37L-8x100 Pelleting: speed (g) 130000 Characterization: Protein analysis Protein Concentration Method Other Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ Alix Not detected contaminants tubulin-alfa/ integrin-beta/ casein-beta Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 117 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2000000000

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EV-TRACK ID	EV210145
species	Homo sapiens
sample type	human milk
condition	Control condition
separation protocol	(d)(U)C
Exp. nr.	1
EV-METRIC %	56