Beckman optima xpn 80

Exosome-free medium was generated by centrifugation at 100,000 g overnight. Naive OT-I cells were seeded and incubated for 1 or 3 days, and then the cell supernatants were harvested for exosome purification. Briefly, cells were centrifuged at 300 g for 5 min remove cells and followed by 2,000 g for another 30 min to remove debris. The supernatant was collected and filtered through a 0.22 μM filter (Corning, NY). Exosomes were precipitated by PEG6000 (Millipore, Darmstadt, Germany) overnight and pelleted by ultracentrifugation at 100,000 g twice for 70 min at 4°C (Beckman Optima XPN-80, Beckman Coulter, Indianapolis, IN). The pellets were collected and washed with cold 1XPBS and followed by ultracentrifugation at 100,000 g twice for 70 min at 4°C (Beckman Optima XPN-80, Beckman Coulter, Indianapolis, IN). Purified exosomes were examined for protein concentrations by Commassie plus Protein Assay Reagent (Thermo Scientific, Rockford, IL) and stored at −80°C until use. Size distribution of exosomes was estimated by a Malvern Zetasizer Nano ZS90 (Malvern, UK) (48 (link)).