Exosome Isolation and Characterization

Exosomes were isolated utilizing a combination of centrifugation, ultracentrifugation, and filtration as we have previously described [16 (link)], or with the Exoquick-TC reagent (System Biosciences, Mountain View, CA, USA) following the manufacturer’s protocol. For ultracentrifugation isolation, conditioned cell culture medium was collected and centrifuged at 10,000 × g for 30 minutes at 4 °C, to remove cells and large debris. The supernatant was filtered using a 0.22-μm pore filter and the exosomes were pelleted at 100,000 × g for 1 h at 4 °C. The exosome pellet was washed with 10 ml of 1 × PBS and pelleted again by centrifugation at 100,000 × g for 1 h at 4 °C. The resulting pellet was either suspended in 1 × PBS for whole exosome applications or further processed for RNA or protein extraction. Plasma exosomes were isolated using the Exoquick reagent (System Biosciences, Mountain View, CA, USA) following the manufacturer’s protocol. The resulting exosome pellet was suspended in PBS and exosome concentration was estimated by Bradford assay.