Polycarbonate centrifuge bottles

The supernatant (50 ml) from virus cultured in Vero cells was collected, and cell debris was removed by centrifugation at 1000 ×g for 15 min at 4 °C. The viruses in the supernatant were concentrated by ultracentrifugation in polycarbonate centrifuge bottles (no. 355603, Beckman Coulter, Brea, CA, USA) using a Beckman L7–65 ultracentrifuge (rotor 70.1 Ti) set at 35,000 rpm for 1.5 h at 4 °C. The resulting pellets were mixed with lysis buffer containing 1% NaCl, 1% sodium dodecyl sulfate (SDS), and 1% Triton-X to produce a virus protein lysate, which was then processed for mass spectrometric analysis via LC-MS/MS by a MicroToF Q II mass spectrometer (Bruker, Germany) coupled to an Ultimate 3000 nano-LC system (Dionex, Sunnyvale, CA, USA) as previously described [24 (link)].

After freezing and thawing 360 mL conditioned media were centrifuged at 6,000 × g in 500 mL conical centrifuge flasks (Beckman Coulter) in an Avanti J-26 XP centrifuge using the swing-out rotor JS-5.3 (Beckman Coulter). Obtained supernatants were transferred into new 500 mL conical centrifuge flasks and supplemented to a final concentration of 10% PEG 6000 and 75 mM NaCl. After overnight incubation at 4°C, EVs were precipitated at 1,500 × g in an Avanti J-26 XP centrifuge using the swing-out rotor JS-5.3 (Beckman Coulter) for 30 min at 4°C. Pellets were re-suspended in 60 mL 0.9% NaCl and transferred in 70 mL polycarbonate centrifuge bottles (Beckman Coulter). Fractions were precipitated at 110,000 × g for 2 h at 4°C in an Optima L7-65 ultracentrifuge using the tight angle rotor Ti45 (k factor 244, Beckman Coulter). Obtained pellets were re-suspended in 1 mL 0.9% NaCl and stored at −80°C until usage.