Isolation of Extracellular Vesicles from Ovarian Cancer Cells

EVs were collected from the supernatant of ovarian cancer cells cultivated in EV-depleted medium (overnight centrifugation at 100,000 x g) using sequential ultracentrifugation 32 (link). The centrifugation protocol included consecutive pre-centrifugation steps at 300 x g (10 min), 2,000 x g (10 min) and 3,500 x g (20 min) for clearance of cells and cellular debris before ultracentrifugation at 10,000 x g (60 min) and/or 100,000 x g (90 min) using an SW41Ti or Type 45Ti rotor for at least twice with resuspension in PBS or HBSS. The amount of EV protein was quantified by Nanodrop 1000 (Thermo Scientific, Schwerte, Germany) and/or using a BCA assay (Pierce). The number of particles was determined by Nanoparticle Tracking Analysis (Particle Metrix).