Sodium chloride (nacl)

10 mL conditioned media (freshly harvested or frozen and thawed) were centrifuged at 10,000 × g in a 5810R centrifuge (Eppendorf). Obtained supernatants were supplemented with 50% w/v stock solutions of PEG 6000, 8000 or 20000 (Sigma-Aldrich, Taufkirchen, Germany) to final concentrations of 6, 8, 10, 12 and 15% PEG and with 3.75 M NaCl (Sigma-Aldrich) to final concentration of 75 mM NaCl (calculated to 15 mL). Samples were mixed gently by inverting the tubes three times. Unless indicated otherwise, samples were stored at 4°C for up to 14 h (overnight). EVs were concentrated by centrifugation at 1,500 × g for 30 min at 4°C in a 5810R centrifuge (Eppendorf, Hamburg, Germany). Supernatants were removed and pellets re-suspended in 250 µL PBS for initial experiments and 1 mL PBS for the comparison of different methods or 250 µL 0.9% NaCl by pipetting, respectively. To remove residues of PEG from the suspension, the EV-enriched fractions were washed with 11 mL PBS or 10 mL 0.9% NaCl, respectively, and centrifuged at 110,000 × g for 2 h at 4°C in an Optima L7-65 ultracentrifuge using the swing out rotor SW40 (k factor 299, Beckman Coulter). The resulting pellet was re-suspended in 250 µL/1 mL PBS or 250 µL 0.9% NaCl, respectively. Obtained EV samples were kept on −20°C for short or −80°C for long-term storage.