Type 70.1 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.

Cells grown to 70% confluence in T175 flasks were washed twice with phosphate-buffered saline (PBS) before FBS-free media was added. The cells were then incubated for 48h before conditioned media was collected for sEV isolation. sEV were isolated by differential centrifugation and ultrafiltration as previously published [16 (link)]. Briefly, cell conditioned media or plasma were centrifuged at 800× g for 10 min, 2000× g for 10 min at 4 °C, and 12,000× g for 10 min at 4 °C to remove cells and debris. The resultant supernatant was centrifuged at 100,000× g for 2 h at 4 °C with the Type 70.1 Ti fixed angle rotor (Beckman Coulter, CA, USA) to pellet the sEVs. The resultant pellet was resuspended in 10 mL PBS, filtered through a 100 kDa Amicon^® Ultra-15 Centrifugal Filter Units (MERCK, Bayswater, VIC, Australia), and centrifuged at 4000× g for 30 min. The sEVs were collected and stored at −80 °C for further experiments. sEVs were characterized according to the recommendations of the International Society of Extracellular Vesicles, by size distribution, abundance of proteins (i.e., CD63 and TSG101) associated with sEVs, and by a morphological assessment involving Nanoparticle Tracking Evaluation (NTA), Western blot, and electron microscopy [37 (link)].

EVs were isolated from the conditioned media following our earlier published method [2 (link)]. Briefly, LNCaP cells were cultured for 48 hrs; thereafter, media was replaced with RPMI1640 supplemented with 10% exosome-depleted FBS and cultured under normoxic (21% O₂) or hypoxic (1% O₂) conditions for 48 hrs. Subsequently, conditioned media was harvested and EVs were isolated by traditional methods using serial centrifugation at low speed, followed by ultracentrifugation (L-80 Ultracentrifuge, Beckman Coulter) at 30,000 rpm using type 70.1 Ti fixed angle rotor (Beckman Coulter). The EVs were also isolated by a precipitation method using commercially available Exoquick^TM reagent (System Biosciences) according to the vendor's instructions. Briefly, conditioned media was overnight incubated with Exoquick^TM reagent, centrifuged at 5,000 rpm for 2 hrs and the pellet was washed once with PBS, and pelleted EVs were resuspended in PBS and stored at −20°C until further use. EVs collected from normoxic and hypoxic PCA cells conditioned media were labelled as EV^Normoxic and EV^Hypoxic, respectively.