Polyethersulfone membrane filter

For EV isolation, A549 and H1299 adherent BCCs were seeded in 150 mm dishes with growth media supplemented with 10% EV-depleted FBS (System Biosciences) and CSCs were seeded in T75 low adherence flask with EV-free CSC media (serum-free DMEM-F12 medium; Gibco-Invitrogen) containing 0.4% Albumin Bovine Fraction V (Sigma-Aldrich), B-27 Supplement (Gibco-Invitrogen), 20 μg/mL EGF (PeproTech), and 10 μg/mL bFGF (PeproTech). The media was collected after 48 hours and centrifuged at 300 × g for 10 minutes at 4°C. The supernatant was transferred to a new polypropylene tube and centrifuged at 2,000 × g for 20 minutes at 4°C. The supernatant then was filtered through a 0.22 μm polyethersulfone membrane filter (Corning Incorporated) and transferred to a polyallomer ultracentrifuge tube and centrifuged at 10,000 × g for 30 minutes at 4°C (Beckman Coultier Optima Max Ultra). The supernatant was then transferred to a new polyallomer ultracentrifuge tube and centrifuged at 100,000 × g for 70 minutes at 4°C (Beckman Coultier Optima Max Ultra). The pellet was then suspended in 1X PBS, and the ultracentrifugation step was repeated to obtain an EV pellet that was suspended in 200 μL of sucrose buffer (5% sucrose, 50 mmol/L Tris, and 2 mmol/L MgCl).