Interferon-? Inhibits Ebola Virus Infection

Rhein, Bethany A.; Powers, Linda S.; Rogers, Kai; Anantpadma, Manu; Singh, Brajesh K.; Sakurai, Yasuteru; Bair, Thomas; Miller-Hunt, Catherine; Sinn, Patrick; Davey, Robert A.; Monick, Martha M.; Maury, Wendy

Isolation of exosomes. The day before exosome collection the culture medium of HEK 293T cells was replaced with DMEM supplemented with pre-spun FBS. To avoid contamination of FBS derived exosomes, FBS were spun at 100,000 x g for 2 hours. The culture supernatants from the HEK 293T cells were collected and the exosomes were isolated from the supernatant by differential centrifugation as described previously [95, 96] . In brief, the cell supernatants were cleared of intact cells and cell debris by centrifugation at 1000 x g for 10 minutes and 10000 x g for 30 minutes at 4°C, and then supernatant was subjected to filtration through a 0.22 μm Amicon filter. The supernatants were concentrated in a 100 kDa MWCO Centricon Plus-15 (Millipore) to 1-2 mL. The concentrated supernatants were spun at 100,000 x g for 70 minutes in TLA 100.3 rotor (Beckman Coulter) to pellet exosomes. The collected exosomes were washed twice in PBS. The recovery of exosomes was estimated by measuring the protein concentration using Bradford assay.