GFP expression constitutes an important tool for the study of stem cells in vitro and in vivo. The results confirm that mDPSC have properties that effectively define them as stem cells. Specific-pathogen-free,

8-week-old male enhanced green fluorescent protein (EGFP) transgenic C57BL/6 mice were maintained at the animal facilities at the Gonçalo Moniz Research Center-FIOCRUZ, Salvador, Bahia, Brazil, and provided with rodent diet and FDA approval PARP inhibitor water ad libitum. The present study was approved by the Institution’s Animal Ethics Committee. The incisors teeth were dissected carefully from the mandibles of male EGFP transgenic C57BL/6 mice after removal of the heads under deep anaesthesia in the CO2 chamber. Special care was taken to avoid contamination by adjacent tissues. Whole dental pulp tissue was gently collected with the aid of a stereotactic microscope (Olympus, Tokyo, Japan), washed three times with sterile saline, and transferred into 24-well plates (Nunc A/S, Roskilde, Denmark). The growth medium consisted of Dulbecco’s Modified Eagle Medium – DMEM medium supplemented with 10%

foetal bovine serum (FBS; Cultilab, Campinas, SP, Brazil), BYL719 mouse 23.8 mM sodium bicarbonate (Sigma, St. Louis, MO, USA), 10 mM Hepes (Santa Cruz Biotechnology, Santa Cruz, CA, USA), 1 mM sodium pyruvate (Sigma), 2 mM l-glutamine (Sigma), 0.05 mM 2-mercaptoethanol (Sigma), 50 μg/ml gentamycin (Sigma), and incubated at 37 °C with 5% CO2. Pieces of tissue explant were used to isolate mDPSC. Culture medium was replaced every 3–4 days. After confluence (usually after 15–20 days), the adherent cells were released with 0.25% trypsin solution (Invitrogen/Molecular

Probes, Eugene, OR, USA) and re-plated (passages) or used in experimental assays, as described below. For cryopreservation, cells were centrifuged and the pellet was resuspended in DMEM medium supplemented with 10% FBS and 10% dimethylsulfoxide (Sigma). Aliquots (5 × 106 cells/ml) were transferred to cryogenic tubes and cooled slowly until −80 °C and, after 24 h, the cryotubes were transferred to a liquid nitrogen container for long-term storage. Cells of the same selleck screening library isolate in different passages were used in the experiments. Cytogenetic analysis of mDPSC metaphases was taken in the 1st and 5th passages, after expansion in the growth medium supplemented with 10% FBS (Cultilab). Cells undergoing active cell division were blocked at metaphase by 0.3 μg/mL colcemid (Cultilab), detached from the growth surface by 0.25% trypsin solution (Invitrogen), and subsequently swollen by exposure to 0.075 M KCl hypotonic solution (Merck). The cells were then fixed in methanol/acetic acid solution (3:1) for slide preparation. Chromosomal analysis of metaphases cells was performed by G banding.