Assessment of cell viability and proliferation using the MTT assay

The MTT assay was used to assess the proliferation of osteoblasts treated with different concentrations (10⁻⁴, 10⁻⁶, 10⁻⁸, 10⁻¹⁰, 10⁻¹², and 10⁻¹⁴ M) of 1,25-dihydroxyvitamin D₃ [14,15]. Formazan synthesized within the cells was suspended in DMSO, and the proliferation rate was compared by assessing its relative optical density (OD). First, we plated 1×10⁴ cells/well on a 96-well plate and allowed them to grow for 3 days. Different concentrations of 1,25-dihydroxyvitamin D₃ were added to the cells when they were 70% confluent, followed by cell culture. Cells in the control group were cultured only in the growth medium, while the other conditions remained constant. On days 1 and 3 of cell culture, 20 µL of 5 mg/mL MTT solution was added to each well, and the medium was removed after 4 hours. Next, 200 µL of DMSO was added to dissolve the synthesized insoluble formazan crystals. The samples were incubated at room temperature for 15 minutes, and OD at 540 nm was measured using a microplate reader (Biotek Instruments, Seoul, Korea). This value of OD represented the amount of MTT reduction by the cells, and was proportional to the number of viable cells in each well. The cell proliferation rates were represented as a percentage value (OD of MTT solution/OD of the control group).