Ab92726

For Nanosight nanoparticle tracking analysis (NTA), 20 μg EVs were dissolved in 1 mL PBS for 1 min to retain the uniform distribution of EVs. Then, EV particle size distribution was directly measured using a Nanosight nanoparticle tracking analyzer (Malvern Panalytical, UK).
For transmission electron microscopy (TEM), 20 μL ultracentrifuged EVs were loaded to carbon-coated copper electron microscope grids for 2 min, followed by negatively 5-min of staining with phosphotungstic acid solution (12501-23-4, Sigma-Aldrich). The grid was then washed with PBS three times to eliminate excess dye solution and kept semi-dry with filter paper. The images were observed using a TEM (H7650, Hitachi, Tokyo, Japan) at 80 kV.
The surface markers of EVs were identified using western blot. EV suspension was concentrated, and then, total protein concentration was examined using a bicinchoninic acid (BCA) assay kit (23227, Thermo Fisher Scientific). SDS-PAGE gel was prepared and protein denaturation and electrophoresis were performed. Afterward, the expressions of EV specific markers CD9 (ab92726, Abcam), CD81 (ab92726, Abcam), Alix (ab76608, Abcam), and calnexin (ab22595, Abcam) were detected.

Exosomes were purified from ADSCs as previously described [25 (link)]. In brief, the medium of ADSCs culture medium was centrifuged under 500×g for 10 min to remove cells and cell fragments were removed at 12,000×g for 20 min. After filtered by a 0.22-μm filter, ADSCs-conditioned medium was harvested. Then, the supernatant fluid was centrifuged at 100,000×g for 30 min to obtain a concentrated liquor of ADSCs-derived exosomes. After centrifugation at 100,000×g for 120 min, the lower liquid layer was diluted by PBS and then centrifuged at 1000×g for 30 min. After washing three times in PBS, the resulting exosomes pellets were resuspended in PBS and frozen at − 80 °C for the experiments. The exosomes were imaged by transmission electron microscopy (JEM-200EX TEM, Tokyo, Japan). For absolute size distribution analysis, the exosomes were diluted to 500 ng/ml and then analyzed by Nanoplus (Gerbrunn, Germany). The proteins in exosomes were analyzed by western blotting using antibody against CD9 (ab92726, Abcam), GM130 (sc71165, Santa Cruz), TSG101(ab83, Abcam).

Exosomes were precipitated by using an exosome precipitation solution (Exo-Quick; System Biosciences) following the manufacturer's instructions [19 (link)]. Ultrastructure and size distribution of the purified exosomes were analyzed by transmission electron microscopy (TEM) and NanoSight (Malvern), respectively. Protein markers, CD9 (Ab92726, Abcam, UK) and CD63 (Ab134045, Abcam, UK), were determined by immune blotting.