Isolation and Characterization of Exosomes from BMMSCs

According to previous methods [24 (link)], exosomes were separated using differential centrifugation. BMMSCs or HO-1/BMMSCs were cultured in exosome-free FBS media (Sesh-biotech, Shanghai, China) for 72 h, and the supernatant was collected and centrifuged at 250×g for 15 min and 3000×g for 30 min, consecutively, to remove cell fragments. After 0.22 μm filtration and centrifugation at 110,000×g for 140 min at 4 ℃, the exosomes were deposited at the bottom of the ultracentrifuge tube. The exosomes were re-suspended in sterile phosphate-buffered saline (PBS), and the supernatant was retained after centrifugation at 10,000×g for 5 min. The obtained exosomes were temporarily stored at − 80 ℃. Western blotting was used to detect exosomes markers, and antibodies against CD9, tumor susceptibility 101 (TSG101), CD63, Calnexin (all ProteinTech, Wuhan, China), and CD81 (Abcam, Cambridge, UK) were used. The morphology of the exosomes was observed using transmission electron microscopy (TEM, Hitachi-HT7700, Tokyo, Japan). The particle concentration, size, and distribution were detected using nanoparticle tracking analysis (NTA, Malvern-NS300, Malvern, UK).