Sw40 swing out rotor

10 mL conditioned media (freshly harvested or frozen and thawed) were centrifuged at 10,000 × g for 45 min in a 5810R centrifuge (Eppendorf, Hamburg, Germany). Supernatants were transferred to 10 mL Ultra-Clear centrifuge tubes (Beckman Coulter), and EVs were precipitated at 110,000 × g for 2 h at 4°C in an Optima L7-65 ultracentrifuge using a SW40 swing out rotor (k-factor 299, Beckman Coulter). For the comparison of the different methods, obtained pellets were re-suspended in 1 mL PBS, for the comparison of the reproducibility in 250 µL 0.9% sodium chloride (NaCl) (Braun, Melsungen, Germany). Fractions were washed with 11 mL PBS or 10 mL 0.9% NaCl, respectively, and re-precipitated by ultracentrifugation exactly as previously performed. Obtained EVs were either processed immediately, kept on −20°C for short-term or −80°C for long-term storage.

10 mL of freshly harvested conditioned media were centrifuged at 110,000 × g for 2 h at 4°C in an Optima L7-65 ultracentrifuge using a SW40 swing-out rotor (k factor 299, Beckman Coulter). Obtained pellets were re-suspended in 1 mL phosphate-buffered saline (PBS; Life Technologies). Obtained EVs were either processed immediately, kept on −20°C for short-term or −80°C for long-term storage.