Isolation of Small Extracellular Vesicles from Prostate Cancer Cells

As published previously,^{62 (link)} for sEV isolation, PrCa cells (PC3 and DU145) were plated in 150 mm cell culture dishes (ThermoScientific, 130183) in their respective complete medium, as described above. After 48 hours of incubation at 37°C, cells were washed with PBS and incubated with serum-free medium (complete media devoid of FBS) for the next 48 hours. The sEVs were isolated by high-speed differential ultracentrifugation of the supernatant (SN) collected after 48 hours of serum-starvation. The dead cells and cell debris were spun down from SN at 2000 xg, 4°C for 20 minutes. The SN collected was spun at 10,000 xg, 4°C for 35 minutes. Next, the SN collected without disturbing the 10,000 xg pellet was spun at 100,000 xg, 4°C for 70 minutes; the pellet was washed in 40 mL PBS followed by a second spin at 100,000 xg, 4°C for 70 minutes. The 10,000 xg and 100,000 xg centrifugation were done in a Beckman Type 45Ti rotor using a Beckman L8-70M  Ultracentrifuge. The final sEV pellets were resuspended in 100 μL PBS.