Exosome Isolation from Cell Culture

After 48 h, we collected the culture medium from the bottom wells in order to determine the amount of exosomes that could have passed through the membrane filter during the incubation period. The media collected from the bottom wells (6 ml of media total, from 3 identical wells) with or without addition of exosomes were collected and subjected to exosome isolation [30 (link)]. Briefly, the media were transferred to 15 ml Beckman tubes (Beckman Coulter, Indianapolis, IN; catalog number #342082) and centrifuged at 2000 g for 30 min to sediment contaminating cells. Clean cell-free media (6 ml each) were transferred into fresh 15 ml centrifuge tubes and 3 ml of exosome isolation reagent was added (Invitrogen, Carlsbad, CA; catalog number 4478359) and mixed by inverting the tubes three times. Exosomes were precipitated by incubating the mix overnight at 4 °C and collected by centrifuge at 10,000 g for 1 h at 4 °C. Supernatant was removed by aspiration and the exosome pellet was suspended in 100 μl of PBS and stored at −80 °C until use.