Surespin 630 swinging bucket rotor

Cells were cultured in DMEM containing 10% FBS, 2 mM L-glutamine, 50 units/ml penicillin and 50 units/ml streptomycin, until 80% confluent. Cells were washed 3 times with PBS and then cultured for 48 h in serum-free DMEM. The conditioned medium was collected and centrifuged for 10 min at 300× g to remove cellular debris. The supernatant was next centrifuged at 3,000× g for 15 min before being filtered through a 0.22 µm polyether-sulfone filter (Nalgene, Rochester, NY, USA) to remove larger vesicles. The filtrate was concentrated approximately 300-fold with a 100,000 molecular-weight cut-off Centricon Plus-70 concentrator (Millipore, Darmstadt, Germany). The concentrated filtrate was centrifuged at 165,000× g in a SureSpin-630 swinging-bucket rotor (Thermo Fisher, Waltham, MA, USA, 30,000 rpm, effective k factor of 219 with 36 ml ultracentrifugation tubes filled to capacity) for 6 h to pellet exosomes. The exosome-enriched pellet was resuspended in 1 ml of PBS containing 25 mM HEPES pH 7.35 (PBS-H) by successive syringing through 22-, 27- and 30-gauge needles, 7 times each. The pellet was washed by centrifuging at 165,000× g for 6 h. The wash steps were repeated until no trace of phenol red was detectable. The final pellet was resuspended in 750 µl of PBS-H, and the protein concentration was determined with a MicroBCA kit (Pierce, Waltham, MA, USA).