Exosome Isolation from Plasma

Peripheral blood samples (6 ml) were collected in EDTA tubes. After centrifugation at 3,000 × g at 4°C for 15 min, at least 3 ml plasma was collected and stored at −80°C. After thawing at 37°C, the plasma was centrifuged at 2,000 × g for 20 min, and the supernatant was obtained. The debris was centrifuged again at 10,000 × g for 20 min. Then, Total Exosome Isolation (from plasma) reagent (Invitrogen, 4484450, California, United States) (Lobb, et al., 2015 (link)) was used according to the manufacturer’s instructions. Briefly, protease K (0.05 times the volume of plasma) was added to the plasma and incubated at 37°C for 10 min. Then, 0.2 volumes of exosome extraction reagent were added to the mixture and incubated at 4°C for 30 min. The mixture was centrifuged at 10,000 × g at room temperature for 5 min. The exosomes, contained in a pellet at the bottom of the tube, were resuspended in PBS and stored at −80°C.