KUSA-A1 mouse osteoblastic cell line was obtained from JCRB Cell Bank (Japanese Collection of Research Bioresources Cell Bank; JCRB1119). Primary periodontal ligament cells were isolated and cloned as described previously (41). Cells were maintained in α-minimum essential media (Wako Pure Chemical Industries, Japan) containing 10% fetal bovine serum (Thermo Fisher Scientific, MA, USA) and kanamycin (60 μg/ml, Wako Pure Chemical Industries) under normal cell culture conditions (37°C, 5% CO2). For osteogenic differentiation, the cells were cultured in the same media supplemented with l-ascorbic acid (50 μg/ml) (013-19641, Wako Pure Chemical Industries) and 10 mM β-glycerophosphate (193-02041, Wako Pure Chemical Industries). Calcein [C001, Dojindo, Japan; stock solution (1 mg/ml) was prepared by dilution from stock (50 mg/ml) in 1 M KOH] was incubated at 1 μg/ml for Ca2+ imaging during culture with normal or osteogenic media. For mineral staining, cells were fixed with cold 100% ethanol for 5 min and stained with 1.0% Alizarin Red S solution (pH adjusted to 6.4; Wako Pure Chemical Industries, Japan).