Cd63 mouse monoclonal antibody

The following antibodies from eBioscience were used for FCM: FITC-anti-mouse-CD41, APC-anti-mouse-CD42, PE-anti-mouse-CD71, PE-anti-human-CD34, APC-anti-human-CD61, FITC-anti-mouse-CD34 and PE/FITC- anti-human-CD41a. A rat anti-mouse-CD41 monoclonal antibody (Abcam) diluted 1:50 in PBS was used for immunohistochemical staining. CD63 mouse monoclonal antibody (Santa Cruz Biotechnology), RHOB monoclonal antibody (ABclonal) and GAPDH monoclonal antibody (Cell Signaling Technology) were diluted 1:1000 and used for Western blotting, and 1 μg per mL human TPO antibody or human TPO receptor antibody (R&D Systems) was used to neutralize TPO in PMPs. The information on the antibodies used in our study is provided in Supplementary Table 2.

Salivary exosomes were isolated by ultracentrifugation. The hamster saliva was centrifuged at 900× g for 15 min to remove debris. The supernatant was sequentially filtrated through a 0.45 μm filter at 14,000× g for 40 min with a 100 K microsep advance centrifugal device (PALL Life Science, New York, NY, USA) at 14,000× g for 40 min. The flow-through was collected and ultra-centrifuged using the Optima L-90K system at 200,000× g at 4 °C for 16 h with an SW41 Ti rotor (Beckman Coulter, CA, USA). The exosome pellet at the bottom of the ultracentrifuge tube was then lysed with 20 μL RIPA buffer (Millipore, MA, USA). The samples were frozen overnight at −20 °C and centrifuged at 13,800× g for 20 min. The supernatant was collected, and the protein concentration was determined using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). Protein samples (5 μg) were separated by SDS-PAGE until a straight-line pattern was observed in the stacking gel. The gel was then stained with 0.5% Coomassie blue. The bands on the gel were cut out, transferred to a 1.5 mL centrifuge tube, and stored at 4 °C for further proteomic analyses. The CD63 biomarkers of the salivary exosomes were detected (Supplementary Figure S1) using CD63 mouse monoclonal antibody (Santa Cruz Biotechnology, TX, USA).