Cells were cultured in media supplemented with 10% exosome-depleted fetal bovine serum (FBS, Hyclone). FBS was depleted of bovine exosomes by ultracentrifugation at 100,000 × g for 70 min. Supernatant fractions collected from 48–72 h cell cultures were pelleted by centrifugation at 500 × g for 10 min. The supernatant was centrifuged at 20,000 × g for 20 min. Exosomes were then harvested by centrifugation at 100,000 × g for 70 min. The exosome pellet was resuspended in 20 ml of PBS and collected by ultracentrifugation at 100,000 × g for 70 min (Sorvall Surespin 630 rotor). Circulating exosomes were isolated from mouse and human plasma as above with an additional filtration through 1.2 μm nylon filters (GE) before the last step of ultracentrifugation. For centrifugation onto sucrose cushions, the samples were diluted 1:10 in PBS after centrifugation at 20,000 × g and then collected by ultracentrifugation (100,000 × g for 70 min) on a 40% sucrose cushion. The floating exosome fraction was collected again by ultracentrifugation as above, and the final pellet was resuspended in PBS. For retrospective studies using frozen plasma, 2 ml of cell-free plasma were centrifuged at 500 × g for 10 minutes, then the supernatant was centrifuged at 20,000 × g for 20 min. Exosomes were then harvested by centrifugation at 100,000 × g for 70 min. The exosome pellet was resuspended in PBS and collected by ultracentrifugation at 100,000 × g for 70 min (Sorvall S100AT5 rotor). The LM10 nanoparticle characterization system (NanoSight) equipped with a blue laser (405 nm) was used for real-time characterization of the vesicles.