SCAP-Exo were isolated according to previous protocols [17 (link), 18 (link)]. For exosome isolation, a conventional culture medium was replaced with a serum-free medium when cells reached 60–80% confluence, and SCAP were cultured for an additional 48 h. Then, the supernatant was centrifuged sequentially at 4°C 3,000 × g for 20 min, 20,000 × g for 30 min, and 120,000 × g for 2 h. Finally, the exosome pellets were resuspended in 200 μL of PBS. 20 μL SCAP-Exo was added to 30 μL lysis buffer (Beyotime Biotech Co., Shanghai, China) for 1 h on ice. The total concentration of exosomes was detected by the bicinchoninic acid (BCA) kit (Beyotime, China). SCAP-Exo was stored at -80°C for subsequent experiments.
The morphology of SCAP-Exo was identified with a transmission electron microscope (H-800, Hitachi, Japan). The size of these exosomes was analysed by nanoparticle tracking analysis. Furthermore, CD9 and Alix were detected by western blot using specific antibodies against CD9 (1 : 250, Abcam, USA) and Alix (1 : 500, Abcam, USA).
The morphology of SCAP-Exo was identified with a transmission electron microscope (H-800, Hitachi, Japan). The size of these exosomes was analysed by nanoparticle tracking analysis. Furthermore, CD9 and Alix were detected by western blot using specific antibodies against CD9 (1 : 250, Abcam, USA) and Alix (1 : 500, Abcam, USA).
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