Isolation of Extracellular Vesicles from Hypoxic and Normoxic Prostate Cancer Cells

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.