X 14r centrifuge

Exosomes were isolated as described previously⁴⁵ . In brief, cell lines were cultured until 70% confluence, washed once by DPBS, and incubated with fresh serum-free medium for 30min at 37°C and 5% CO₂. After 30 minutes, the medium was replaced either by serum-free medium (SFM) or exosome-free serum containing medium (EFM) and returned to the incubator for the indicated exosome production period. The cell-conditioned media were collected at different time points and exosomes were isolated by differential centrifugation as follows: 300 ×g for 10 minutes to remove cells, 2,600 ×g for 10 minutes to remove residual cells and debris, 10,000 ×g for 60 minutes to remove microvesicles, and 100,000 × g for 2 hours to collect nano-scaled vesicles in pellets. The resulting pellet was resuspended, washed once in DMEM, and repelleted at 100,000 ×g for 2 hours. Differential centrifugation was conducted using a Beckman Coulter X-14R centrifuge and a Beckman Coulter XL90 ultracentrifuge with proper rotors, open-top (Cat#: 355631) or capped (Cat#: 355618, Cat#: 355655) thickwall polycarbonate tubes (Beckman Coulter). Once isolated, nano-scaled vesicles were resuspended in DPBS and kept on ice.