Exosome isolation using OptiPrep™ density gradient medium

MDCK, MDCK^YBX1, and 21D1 cells (50x 150 mm dishes per cell line) were cultured to 70% confluence in DMEM+10% FBS, washed three times with DMEM, and cultured in DMEM for a further 24 h. Conditioned media (CM) was collected (∼750 mL) and centrifuged (480 x g for 5 min, 2000 x g for 10 min) as described [24]. Supernatants were centrifuged at 10,000 x g for 30 min at 4°C, and the resulting supernatants at 100,000 x g for 1 h to isolate crude exosomes [22, 38, 39]. Exosomes were washed in PBS, and layered onto OptiPrep™ density gradients for ultracentrifugation at 100,000 xg for 18 h, as previously described [40]. Twelve individual 1 mL fractions were collected (from top of the gradient with increasing density) and each fraction diluted with 2 mL PBS. After centrifugation at 100,000 x g for 1 h at 4°C, supernatants were discarded and pellets washed with 1 mL PBS and resuspended in 50 ml of PBS for downstream analysis [38].