Pvdf membrane syringe filter

The human prostate cancer cell line PC3 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). PC3 cells were maintained in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS) (VWR, Radnor, PA, USA) and 5 U/ml Penicillin Streptomycin (Thermo Fisher Scientific). For EV separation, cells were grown in medium containing 10% exosome‐depleted FBS (Thermo Fisher Scientific) until they reached a confluency of ∼90% (after approximately 48 h). The cell number at CCM harvest was around 7.5×10⁶/T150 flask. Each flask was used to condition 20 ml of culture media. Ten to fifteen T150 flasks were used for each experiment (the input volume of CCM for each method was different and details can be found in each separation method section). First, the fresh CCM was immediately centrifuged at 1000 × g for 10 min to eliminate cells and large debris. Second, the supernatant was centrifuged at 10,000 × g for 20 min at 4°C to remove small debris, apoptotic bodies and other large EVs. Third, the supernatant was filtered through a 0.45 μm hydrophilic PVDF membrane syringe filter (Thermo Fisher Scientific). The pre‐processed CCM was either used fresh for EV separation or stored at −80°C until use, limiting freeze‐thaw cycles to a maximum of one.

For EV separation from plasma, whole blood from fasted healthy donors was collected in an ethylenediaminetetraacetic acid (EDTA) coated tube (BD Biosciences, San Jose, CA, USA.). Separation of plasma from other components was achieved by centrifugation at 1000 × g for 10 min, after which the plasma was transferred to a new tube. The plasma was further pre‐cleaned by two centrifugations at 2500 × g for 15 min to remove cells, large debris, and thrombocytes with supernatant collected after each spin. The supernatant was then centrifuged at 10,000 × g at 4°C for 20 min to remove apoptotic bodies, other large EVs, and some non‐EVs components. For further reduction of lipoproteins, likely the most abundant source of plasma proteins, the supernatant was filtered using a 0.45 μm hydrophilic PVDF membrane syringe filter (Thermo Fisher Scientific). The plasma was either used freshly for EV separation or stored at −80°C for later use, limiting freeze‐thaw cycles to a maximum of one.