2.3. sEV isolation by differential ultracentrifugation

Conditioned medium was collected 24 h after pulse media exchange or at the indicated chase time intervals and serially centrifuged at 2000 g for 20 min at 4°C to discard cells and large debris. The supernatant was centrifuged at 10,000 g for 20 min at 4°C for microvesicles and other debris. The supernatant was then ultracentrifuged 100,000 g for 90 min at 4°C and the pellet (100K pellet) was washed in 12 mL of PBS and centrifuged again at 100,000 g in an SW41 Ti swinging bucket rotor (Beckman Coulter). Pellets were resuspended in 100–150 µL of PBS.