Exosome Isolation and Characterization from BMSC

Alpha-MEM (Gibco, Green Island) containing 20% FBS (Gibco) was centrifuged for 18 h at 200,000 g to deplete exosomes. BMSCs were cultured in exosome-free medium containing 10% FBS, and BMSCs at passages 4-6 were selected for exosome collection. A total of 2 × 10⁶ MSCs were cultured in 100 mm culture dishes under normal/hypoxic conditions for 72 h, and 10 ml of the supernatant was taken. Exosomes were then isolated from the supernatant after centrifugation^{16 (link)}. Then, exosomes were resuspended in PBS.
For identification of exosomes, samples were evaluated under a JEM-2100 transmission electron microscope (TEM; JEOL, Tokyo, Japan). Images were acquired using the PARTICLEMEIRIX system. Nanoparticle tracking analysis (NTA) was performed using the NanoSight NS300 system (Malvern Instruments, Malvern, UK). The Brownian motion of exosomes in PBS was recorded and tracked, and the size distribution was analyzed using the Stokes-Einstein equation. The exosome characteristics were identified by detecting the expression of the exosome-specific surface markers with rabbit anti-CD63 (1:2000, ab216130, Abcam, UK), rabbit anti-TSG101 (1:10,000, ab125011, Abcam), rabbit anti-CD81 (1:10,000, ab109201, Abcam) and rabbit anti-Calnexin (1:100,000, ab92573, Abcam) antibodies by Western blot analysis.