Exosome Purification and Characterization

The exosomes were purified by sequential centrifugation as previously described [44 (link),62 (link),63 (link)]. In brief, cells were cultured in media supplemented with 10% exosome-free FBS. After 72 h, cells were digested and counted. The supernatants were centrifuged at 500× g at 4 °C for 10 min to remove living cells. To remove dead cells and large cell debris, the supernatants were then centrifuged at 2000× g at 4 °C for 10 min. To remove large vesicles, the supernatants were centrifuged at 10,000× g at 4 °C for 30 min. Finally, the exosomes were harvested by centrifugation at 100,000× g at 4 °C for 70 min (Beckman 32 Ti rotor, Germany). The exosomes were washed with PBS (Thermo, USA) and pelleted again through ultracentrifugation at 100,000× g at 4 °C for 70 min. The exosome pellets were resuspended in the corresponding volume of PBS according to cell numbers to ensure that the same volume of PBS contained exosomes from the same cell number. Exosome size and particle concentration were analyzed using an NS300 nanoparticle characterization system (NanoSight, Malvern Instruments, Malvern, UK) equipped with a red laser (580 nm). The exosomes were lysed with denaturing RIPA buffer (plus the cocktail), and their markers were analyzed by WB. The exosomes were stored short-term at 4 °C and long-term at −80 °C.