EV isolation

To isolate EVs, we followed ultracentrifugation isolation procedures recently reported (Method I) [25]. Cell culture supernatant (15 mL) or serum (1 mL) was centrifuged at 300×g for 10 min at 4 °C to remove any cells and large debris. The EV containing supernatant was collected and centrifuged at 2000×g for 15 min at 4 °C to remove apoptotic bodies and debris. The supernatant was further subjected to 10,000×g for 30 min at 4 °C to remove larger microvesicles. This supernatant was spun twice at 120,000×g for 70 min at 4 °C, with the pellet resuspended in DI water in between spins. The final supernatant was discarded, and the pellet resuspended in MilliQ water or PBS (125–225 μL). This solution was aliquoted and frozen at −80 °C until used. Freeze–thaw cycles were minimized for downstream application. Centrifuge steps for SK-OV-3 samples were completed in an Optima LE-80K centrifuge with a SW-28 rotor. Slow speed centrifuge steps for serum samples were completed a Beckman Coulter Microfuge 20R centrifuge with an FA361.5 Biosafe rotor and slow speed spins were completed in a Beckman Optima TLX Ultracentrifuge with a TLA 100.1 fixed angle rotor.

PMSC EVs followed the same general protocol with two minor differences (Method II). First, after the 2000×g spin two filtration steps were added. The supernatant was transferred to a 0.2 μm filter and vacuum filtered. The EVs in the filtrate were then concentrated in sterilized Centriprep 100,000 K cutoff filters at 8836×g for 30 min, adding additional media until all media had been concentrated, before continuing on to the 10,000×g spin. Finally, the samples were subjected to 120,000×g for 90 min each instead of 70 min during the last two steps. Centrifuge steps for PMSC samples were completed in an L7 Ultracentrifuge with an SW-28 rotor (Beckman Coulter).

All EV samples were aliquoted to minimize freeze–thaw cycles and stored at −80 °C until used.