Exosomes isolation methods
First method is Total Exosome Isolation (TEI), where milk samples centrifuged at 2000 × g for 30 min, then supernatant was collected and 2.5 mL of TTEI reagent was added (1:2 ratio), mixed well until homogeneity and incubated overnight at 4 ºC. The solution was then centrifuged at 10,000 × g for 1 hour at 4 °C. Supernatant was discarded; pellet and sediment exosomes were re-suspended in equal volume of 1 × PBS. The second method, isoelectrical precipitation (IP), which was performed according to Yamauchi M. et all (2018). Briefly, skimmed milk was diluted with distilled water, and pH was adjusted to 4.6. Samples were then centrifuged at 5100 × g for 20 min at 25 ºC, and supernatant was collected and filtered using 0.45 µm filters. The third isolation method was size exclusion chromatography (SEC) and performed according to manufactures instructions. This involves isolating exosomes from the supernatant of cell cultures and complex biological fluids. Since the separation is based on size, the vesicles pass through the column, are retained and eluted in the void. Proteins and other contaminants that are smaller than the pores are retained by the column and eluted later. Isolation using original size exclusion columns removes >99% of background protein contaminants and up to 95% of high-density lipoprotein contaminants from samples in a single isolation. Column qEV original 70 (IZON Science, New Zealand) that has an optimal recovery range from 70 nm to 1000 nm was used in the experiment. Columns were washed by filtered 1 × PBS, followed by a second wash containing 19 mL of filtered 1 × PBS and 0.5 mL of supernatant. A total of 5-10 concentrated fractions were collected for the study.