Cd146 percp cy5

Apical papilla tissue was collected from third molars extracted from medically healthy patients (16 to 25 years old); informed consent had been obtained in accordance with a protocol approved by the Institutional Review Board at the Arthur A. Dugoni School of Dentistry at the University of the Pacific (IRB protocol #16-128). SCAP culture was established as previously described^{45 (link)}. Similarly, periodontal ligament cells (PDLC) were obtained from scraped PDL tissue. Cells were cultured in alpha minimum essential medium (α-MEM) supplemented with 1% L-glutamine, 1% penicillin/streptomycin/amphotericin B (all ThermoFischer Scientific, Pittsburgh, PA, USA) and 10% human serum (HS) (Sigma-Aldrich, St Louis, MO, USA). SCAP and PDLC at passage 4 were analyzed for cell surface antigen expression by flow cytometry using Guava easyCyte 8HT flow cytometer (EMD Millipore, Billerica, MA, USA). Fluorochrome-conjugated monoclonal mouse anti- human against CD45-APC/Cy7, CD90-PE-CY7, CD105-APC, CD146-PerCP/Cy5.5 and STRO-1- FITC or isotype controls (all from BioLegend, San Diego, USA) were used. Data were analyzed using InCyte 2.5 software (EMD Millipore). Cells counts were performed at each passage, and the population doubling times (PDTs) were calculated.