Type 70 ti fixed angle rotor

Technical protocols of 4 EV separation methods compared in this study were schematically diagrammed as shown in Figure S1. Fresh or thawed CCM, plasma, or urine were centrifuged at 120,000 × g for 2 h at 4°C. After the first UC spin, we used PBS to resuspend the EV pellet, followed by a second UC spin in the same tube at 120,000 × g for 2 h at 4°C. For EV separation from CCM and urine (100–150 ml), a Beckman Coulter Type 70 Ti fixed angle rotor was used (adjusted k‐factor 131, maximal acceleration, maximal deceleration). For EV separation from plasma (4 ml), the Beckman Coulter Type 70.1 Ti fixed angle rotor was used (adjusted k‐factor 102, maximal acceleration, maximal deceleration). After the removal of supernatant, the EV pellets were resuspended and collected in 100 μl PBS.

Hs578Ts(i)₈ cells were seeded at 5 × 10⁵ cells per T175 flask. The following day the medium was replaced with DMEM medium supplemented with 10% dFBS and 1% penicillin/streptomycin and the cells were cultured for another 5 days. EVs were collected by differential ultracentrifugation, adapted from a previously published protocol [4 (link)]. Specifically, CM was collected and the cell number was counted. The CM was centrifuged at 300 g for 10 min at 4 °C, three times, as a pre-clearing step to remove cellular debris. The CM was transferred to new 50ml tubes and centrifuged at 2,000 g for 20 min at 4 °C (producing the 2 K pellet). The CM was transferred to Quickseal 39ml tubes (Beckman coulter, Cat. #:342,414) and centrifuged in a Type 70 Ti fixed angle rotor (Beckman coulter, Cat. #:337,922) at 10,000 g for 30 min (producing the 10 K pellet). The CM was transferred to new Quickseal tube and centrifuged in a Type 70 Ti rotor at 100,000 g for 70 min (producing the 100 K pellet). Finally, the CM was transferred to another Quickseal tube and centrifuged in a 70 Ti rotor at 200,000 g for 65 min (producing the 200 K pellet). All pellets were washed with PBS and re-centrifuged at the same speed as previously, before resuspending each EVs sub-population pellet in 150 µl of PBS and storing in Protein LoBind tubes (Eppendorf, Cat. #: 0030 108.116) at -80 °C.

For the dUC approach, separation of EVs was performed following the protocol described by Théry et al. [12 (link)]. Briefly, 312 mL freshly harvested CM was spun at 300× g for 10 min (5810R centrifuge, Eppendorf, Hamburg, Germany). The resulting supernatant was then centrifuged at 2000× g for 10 min. After any dead cells were removed, the CM was further spun at 10,000× g for 30 min at 4 °C in a 5810R centrifuge. The supernatant was then transferred to 8 × 39 mL Quick-Seal^® polypropylene centrifuge tubes (Beckman Coulter, Brea, CA, USA; Cat. #: 342414) and spun at 100,000× g for 70 min at 4 °C in an Optima XPN-100 Ultracentrifuge using a Type 70 Ti fixed-angle rotor (Beckman Coulter). The resulting eight pellets with EVs were washed in a combined total of 39 mL of 0.9% NaCl (Sigma-Aldrich, Cat. #: 71376) and ultracentrifuged exactly as previously performed. The final EV pellet was resuspended in 100 μL of 10 mM HEPES/NaCl (Thermo Fisher Scientific, Cat. #:15630049) and stored at −80 °C.