Dmem alpha modified eagle s medium

MSCs were cultured in Transwell chambers with an 8-μm pore size membrane (Corning, Costar, MA, USA). The upper chamber was seeded with MSCs (2.0 × 10⁴ cells) which were grown in 100 uL DMEM alpha modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with TNFα (final concentration is 10 ng/ml) [25 (link)–27 (link)] and no fetal bovine serum (FBS; Invitrogen) while there was 600 uL DMEM alpha modified Eagle’s medium with TNFα (10 ng/ml) and 15% FBS in the bottom chamber. After 48 hours, the transferred cell numbers were counted in randomly selected fields using microscopy (OLYMPUS, Japan) at 200× magnification.

Gingival tissues were isolated from C57BL/6 mice. Solutions of 75% ethanol and phosphate-buffered saline (PBS) were used to disinfect and rinse the tissues. After that, gingival tissues were digested by a solution of 3 mg/ml collagenase type I (Sigma-Aldrich, USA) and 4 mg/ml dispase (Sigma-Aldrich, USA) for 1 h at 37 °C. A 70-μm strainer (Falcon, USA) was used to filter dissociated GMSC suspensions. GMSCs were cultivated in a humidified incubator under 5% CO₂ at 37 °C in DMEM alpha modified Eagle’s medium (Invitrogen, USA), renewal with 20% fetal bovine serum (FBS; Invitrogen), 100 μg/ml streptomycin, 100 U/ml penicillin, and 2 mmol/l glutamine (Invitrogen).

SCAPs were gently isolated from the apical papillae of immature third molars obtained from patients in the Beijing Stomatological Hospital of the Capital Medical University. The patients gave an informed consent prior to the study. The apical papilla tissues were first covered in a solution comprising 3 mg/ml collagenase type I (Invitrogen, Carlsbad, CA, USA) and 4 mg/ml dispase (Invitrogen) for 60 min at 37 °C. Single-cell suspensions were then obtained using a 70-mm strainer (Falcon, BD Labware, Franklin Lakes, NJ, USA). The suspensions were incubated in DMEM alpha modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 15% fetal bovine serum (FBS; Invitrogen), 200 mM l-glutamine, and 10,000 units/ml penicillin-streptomycin (Invitrogen) in an incubator set at 37 °C and 5% carbon dioxide. We used flow cytometry to identify stem cells, and the results were showed in our previous article [33 ]. The suspensions were transferred to a fresh cell culture medium every 3 days.
Human embryonic kidney 293T (HEK 293T, American Type Culture Collection, Manassas, VA, USA) cells were incubated in complete DMEM supplemented with 10% FBS (Invitrogen), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). The 293T cells were used to package viral constructs.