Exosome Isolation from Cell Culture

Cell culture supernatants of Huh7, JFH1 infected Huh7 and HCV FL replicon cells were collected and centrifuged at 800× g for 5 minutes to remove cell debris. To concentrate microvesicles in cell culture supernatants, cleared supernatants were then transferred into Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-100 membrane (Millipore, cat. #UFC910024) and followed by series of centrifugations at 3,500× g for 20 minutes. Concentrated culture supernatants were mixed with the appropriate volume (50μl—150μl) of Exoquick (System Bioscience cat. #EXOQ5A-1) according to the manufacturers’ specifications and centrifuged at 6000×g for 20min. The exosome pellet was washed 2 times with PBS by centrifugation at 800x g at 4°C for 5 minutes and re-suspended in 1X phosphate buffer saline (PBS). For JFH1 and serum exosomes from human samples, further positive anti-CD63 immuno-selection was performed as described previously [11 (link)].