Cd81 sc 9158

The antibodies to Tet1 (ab191698), Tet2 (ab94580), P2rX7 (ab48871), CD146 (ab24577), PDGFRα (ab65258), and OCN (ab10911) were purchased from Abcam (Cambridge, MA, USA). Antibodies to ALP (sc-28904), CD9 (sc-9148), and CD81 (sc-9158) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Antibody to Runx2 (8486) was obtained from Cell Signaling Technology (Danvers, MA, USA). Antibody to Tet3 (20602) was purchased from Novus Biologicals (Littleton, CO, USA). Anti-CD34-PE (551387) and SCA1-PE (553108) antibodies were purchased from BD Biosciences (San Jose, CA, USA). Anti-CD105-PE (12-1051-82), CD45-PE (25-0454-82), CD73-PE (12-0739-42), and CD90-PE (15-0902-82) antibodies were purchased from eBioscience (San Diego, CA, USA). Anti-β-Actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cells were washed twice with PBS and then lysed in the RIPA lysis buffer (Sigma-Aldrich Corp.) at 4°C. After centrifugation, soluble proteins were quantified by BCA assay (Beyotime). Equal amounts of protein were separated by 15 Tris-glycine SDS–polyacrylamide gel (Invitrogen) electrophoresis, transferred to a PVDF membrane (Merck Millipore, Billerica, MA, United States), blocked with 5% BSA in PBST (PBS with 0.1% Tween), and incubated overnight at 4°C with primary antibodies targeting GAPDH (CW0100, CWBIO, Beijing, China), CD9 (ab92726, Abcam, Cambridge, United Kingdom), TSG101 (ab125011, Abcam), CD81 (sc-9158, Santa Cruz Biotechnology, Texas, TX, United States), PINK1 (#6946), Parkin (#4211), and LC3-I/II (#12741) (all from Cell Signaling Technology, Danvers, MA, United States). After incubation with the respective secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, United States), the bands were visualized using enhanced chemiluminescence (Pierce, Rockford, IL, United States) and quantified using ImageJ (Media Cybernetics, Silver Springs, MD, United States).

For the transmission electron microscopy (TEM) observation, the exosomes derived from 1 × 10⁶ human DPSCs were suspended in 2.5% glutaraldehyde, loaded onto copper grids, and then examined by TEM at 100 kV (JEOL, Tokyo, Japan).
For Western blot analysis, total proteins of human DPSCs or DPSC-derived exosomes were extracted by a protein extraction kit (RIPA Cocktail; Thermo, Waltham, MA, USA), separated by SDS-PAGE, and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA). After being blocked with 5% bovine serum albumin (BSA) and 0.1% Tween-20 for 1 h, the membranes were incubated with CD9 (sc-9148; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and CD81 (sc-9158; Santa Cruz Biotechnology, Santa Cruz, CA, USA) primary antibodies overnight at 4 °C. The blots were then incubated with HRP-conjugated secondary antibodies for 1 h at room temperature and developed by enhanced chemiluminescence detection. Quantitative analysis was performed with ImageLab software.
For nanoparticle tracking analysis (NTA), the exosomes were diluted uniformly in phosphate-buffered saline (PBS) solution and were further measured by a NanoSight analysis system (Malvern Panalytical, Malvern, England) to analyze the size distribution and concentration.