Isolation of Exosomes from Cell Culture

CCM was harvested from SK-MES-1 cells and centrifuged using a Beckman Coulter Allegra^® X-15R centrifuge at 300 g at 4°C for 10 minutes to remove detached cells. Supernatant was collected and filtered through 0.22 µm filters (Merck Millipore) to remove contaminating apoptotic bodies, microvesicles and cell debris. Clarified CCM was then centrifuged in a Beckman Coulter Optima™ L-80XP Ultracentrifuge at 100,000 g_avg at 4°C for 90 minutes with a Type 50.2 Ti rotor (k-factor: 157.7) to pellet exosomes. The supernatant was carefully removed, and crude exosome-containing pellets were resuspended in 1 mL of ice-cold PBS and pooled. A second round of ultracentrifugation [100,000 g_avg at 4°C for 90 minutes with a Type 50.2 Ti rotor (k-factor: 157.7)] was carried out, and the resulting exosome pellet resuspended in 500 µL of PBS (Supplementary Fig. 1).