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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140100	1/1	Homo sapiens	Milk	(d)(U)C DG Filtration	Torregrosa Paredes P	2014	43%
Study summary Full title Differences in exosome populations in human breast milk in relation to allergic sensitization and lifestyle All authors Torregrosa Paredes P, Gutzeit C, Johansson S, Admyre C, Stenius F, Alm J, Scheynius A, Gabrielsson S Journal Allergy Abstract BACKGROUND: Breast-feeding has many beneficial effects on the developing immune system of the newbor (show more...)BACKGROUND: Breast-feeding has many beneficial effects on the developing immune system of the newborn. Breast milk contains immunoregulatory factors, such as nano-sized vesicles named exosomes. This study aimed at characterizing breast milk exosomes from human early milk and mature milk and to investigate whether allergic sensitization and an anthroposophic lifestyle could influence the exosome profile. METHODS: Breast milk was collected from 22 mothers at day 3-8 and from 61 mothers at 2 months postpartum, all part of the ALADDIN birth cohort. Isolated exosomes were captured on anti-MHC-class II- or anti-CD63 beads and analyzed by flow cytometry. Exosomal phenotype was related to lifestyle and allergic sensitization of the mothers, and sensitization of the child at 2 years of age. RESULTS: We found a higher content of exosomes in early milk compared with mature milk. Early milk exosomes were enriched in HLA-DR molecules and displayed significantly lower levels of HLA-ABC compared with those in mature milk. Phenotypically different subpopulations of exosomes were found in mature milk. Significantly lower levels of MUC1 were detected on CD63-enriched exosomes from sensitized mothers compared with nonsensitized. Furthermore, women with an anthroposophic lifestyle had significantly lower MUC1 expression on their HLA-DR-enriched milk exosomes and up-regulated levels of CD63 on CD63-enriched exosomes compared with nonanthroposophic mothers. Notably, mothers whose children developed sensitization had an increased amount of HLA-ABC on their milk exosomes enriched for CD63. CONCLUSIONS: The phenotype of exosomes in breast milk varies with maternal sensitization and lifestyle, which might influence allergy development in the child. (hide) EV-METRIC 43% (65th 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 Milk Sample origin NAY Focus vesicles exosomes 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 DG Filtration Protein markers EV: CD81/ CD63/ MUC1/ MHC2/ CD36 non-EV: Proteomics no EV density (g/ml) 1.12-1.18 Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens Sample Type Milk Separation Method (Differential) (ultra)centrifugation dUC: 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 Pelleting performed Yes Pelleting: time(min) 90 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2 Orientation Top-down Speed (g) 200000 Filtration steps > 0.45 µm, 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Western Blot Antibody details provided? No Detected EV-associated proteins MHC2/ CD36/ MUC1/ MHC2 ELISA Antibody details provided? No Detected EV-associated proteins MHC2/ CD36/ MUC1/ MHC2 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) Yes Characterization: Particle analysis NTA

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EV-TRACK ID	EV140100
species	Homo sapiens
sample type	Milk
condition	NAY
separation protocol	(d)(U)C DG Filtration
Exp. nr.	1
EV-METRIC %	43