To analyze the effect of TRPM8 channel modulators, osteogenic differentiation was carried out in the presence of Menthol 100 μM, Icilin 10 μM or BCTC 20 μM. Medium (StemPro® Osteogenesis Differentiation Kit) containing the drugs was changed every third day during the differentiation process. Cells incubated with regular media were used as a negative control. Osteogenic cell differentiation was determined by alizarin red staining (as previously described). The differentiated area was calculated with the ImageJ software, and changes in expression of ALPL (Alkaline phosphatase) were measured by qPCR by the comparative CT method. Statistical analyses were performed using GraphPad Prism (One-way ANOVA).

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