Figure 1. Hypoxia increases the release of extracellular vesicles (EV) from pancreatic cancer cells.
(A) Conditioned media from pancreatic cancer cells (MiaPaCa and AsPC1) cultured under normoxia and hypoxia (moderate, 1.0% O2; extreme, 0.1% O2) for 48 h, was collected and total EV isolated by ultracentrifugation (120,000g for 2h). (B) Differential ultracentrifugation was performed to isolate size-based sub-fractions of EV (large, 2000g × 30min; moderate, 16500g × 30min; small 120,000g × 120min). (C) To examine the release kinetics, cells were incubated under normoxia or hypoxia (1.0% O2; 0.1% O2) for 0–48 h and EVs sub-fraction isolated at 12 h time intervals. In all experiments, EV pellets were suspended in equal volume of PBS and EV release estimated by measuring the protein concentration. Data is presented as mean ±S.D. of biological replicates.