Bca quantitation kit

For identification of circulating EVs, EV morphology was analyzed using a transmission electron microscope (TEM, Hitachi, Tokyo, Japan), and a ZetaView PMX 120 (Particle Metrix, Inning am Ammersee, Germany) was used for nanoparticle tracking analysis (NTA) to analyze particle size distribution. Additionally, the specific EV surface markers (CD9, CD63, Calnexin, and TSG101) were measured through Western blotting (WB) assay. The BCA quantitation kit (Beyotime, Shanghai, China) was adopted for determining protein content in EV suspension. Primary antibodies used in this study were as follows: anti-CD63 (1:500; Abcam, Cambridge, Britain), anti-CD9 (1:500; Abcam), anti-TSG101 (1:500; Abcam), and anti-Calnexin (1:500; Abcam). For the in vitro experiments, three independent repeated tests were performed (n = 3).

ADSC‐exos were isolated from cell supernatants as described previously^[56, ^57] When ADSCs reached 80−90% confluence, the FBS‐free culture medium was changed. The culture supernatant at 3000 X g was centrifuged for 30 min to remove dead cells, followed by centrifugation at 10 000 X g for 30 min to eliminate cellular debris. After that, the remaining supernatant was filtered through a 0.22 µm filter to remove vesicles and apoptotic bodies. The clarified supernatant was ultracentrifuged at 100 000 X g for 90 min, and the exosome‐containing pellets were resuspended in PBS. The above operation procedures were performed at 4 °C, and the obtained exosomes were stored at −80 °C or used immediately. The average protein concentration of the exosome suspension was measured by a BCA quantitation kit (Beyotime, China). The particle size distribution and ζ‐potentials of exosomes were measured by dynamic light scattering (DLS). The isolated exosomes were characterized by transmission electron microscopy (TEM) for ultrastructural identification. Western blot was performed to evaluate the expression of ADSC‐exos positive markers CD9 (ABclonal, China), CD63(ABclonal, China), CD80 (Abcam, UK), and TSG101 (HuaBio, China), and the negative marker calnexin (ABclonal, China).