2.1.3 Physical isolation by six-fold differential ultracentrifugation and filtration (the physical 6-dUC procedure)

Based on the protocol described from Izumi et al. (Izumi et al., 2013), a 60-mL sample of commercial goat milk was divided into two 30-mL centrifuge tubes and centrifuged for 10 min at 1,200 × g to remove fat-containing vesicles, cells and large debris. Defatted supernatants were centrifuged twice at 21,500 × g for 30 min to eliminate casein and residual fat. Next, the resultant supernatants were centrifuged again at 21,500 × g for 60 min in order to remove the remaining casein. The milk whey was filtered through 0.65-, 0.45-, and 0.22-µm PES syringe membrane filters to step-wise eliminate residual contaminants. sEVs were precipitated by ultracentrifugation at 100,000 × g for 90 min. Pellets were washed with 5 mL of 1X PBS and ultracentrifuged at 100,000 × g for 90 min. Precipitated sEVs were dispersed in 200 µL of 1X PBS.