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.