Exosome Isolation and Characterization

Exosomes were isolated as described previously39 (link). Briefly, conditioned medium was harvested 48 hours after cell seeding. After centrifugation at 3000 g to remove cellular debris, the supernatant was filtered through 0.22-μm PVDF filter (Millipore) to remove large vesicles. Exoquick Exosome Precipitation Solution (System Biosciences) was added to the filtered culture medium and mixed well. After refrigeration for 12 h, the mixture was centrifuged at 1500 g for 30 min. Exosome pellets were resuspended with PBS. The protein concentrations were measured by the Bradford method using a protein assay kit (Bio-Rad) as previously reported40 (link). To confirm the contents of the purified exosome samples, we performed immunoblotting to detect tetraspanin CD63 (Supplementary Fig. 6A). The size distribution of the exosomes in the culture supernatants was evaluated with the NanoSight LM10 system using Nanoparticle Tracking Analysis (NTA) software v2.3 (NanoSight Ltd, Amesbury, UK). The mean vesicle size in the samples was 89 nm (Supplementary Fig. 6B), in accordance with previous reports35 (link)41 (link). Purified exosomes were added to the culture medium at a concentration of 50 μg/ml. For plasma samples, exosome isolation was performed using ExoQuick Plasma prep and the Exosome precipitation kit (System Biosciences).