Cd146 pe

The cells isolated from PDL tissues were characterized via flow cytometry. Cells (passage 2) were detached from dishes by 0.25% trypsin-EDTA and washed with phosphate-buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA). Then, 50 μL cell suspension that contained 1 × 10⁶ cells were incubated with the conjugated antibody against CD90-PE (Thermo Fisher Scientific, Waltham, MA, USA), CD146-PE (Thermo Fisher Scientific, Waltham, MA, USA), CD34-PE (Thermo Fisher Scientific, Waltham, MA, USA), and CD45-FITC (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min on ice in the dark. Then, the cells were washed twice and resuspended with cold PBS containing 1% bovine serum albumin (BSA, Beyotime, Shanghai, China) and analyzed using a flow cytometry (Accuri C6 Flow Cytometer, BD Bioscience, San Jose, CA, USA). Data were analyzed with Flowjo software (BD Bioscience, San Jose, CA, USA). Cells between passages 3–5 were used for subsequent experiments.

Cell surface marker expression was evaluated using the following primary antibodies: the stage-specific embryonic antigen 4-PE (SSEA4-PE; BioLegend, Dedham, MA, USA), CD73-APC (Thermo Fisher Scientific), CD90-APC-Vio770 (Miltenyi Biotec, San Diego, CA, USA), CD105-PerCP-Vio700 (Miltenyi Biotec), and CD146-PE (Thermo Fisher Scientific). Expanded cells (2 × 10⁵ each group) were first blocked through a 30 min incubation in cold phosphate-buffered saline (PBS) containing 0.1% ChromPure Human IgG whole molecule (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) followed by incubation with primary antibodies at 4 °C for 30 min. Fluorescence was evaluated by a FACS Calibur (BD Biosciences) using the FCS Express software package (De Novo Software).