2.5. Cell Culture and Pharmacological Treatment with Calcium Ionophore, A23187

For the pharmacological treatment, 7 × 10⁵ skin fibroblasts were seeded in each well of 4-well plates (21.8 cm²/well). Cells were incubated with A23187, a calcium ionophore (Sigma-Aldrich, Darmstadt, Germany). Each cell preparation was treated with two doses of A23187, 1 µM and 10 µM, in duplicates and at three different times of incubation (4, 24, and 48 h). Skin fibroblasts were deprived of FBS and antibiotics 24 h before treatment. A23187 was solubilized in DMSO and diluted in FBS-free DMEM before its addition to cells. In control wells, a similar volume of DMSO, diluted in FBS-free DMEM, was added. Expression levels of FOS gene were quantified as a positive control for the entry of calcium in the cells [8].