Effect of 1,25-dihydroxyvitamin D₃ on spontaneous calcium responses in rat dental epithelial SF2 cells revealed by long-term imaging

Genetically encoded calcium indicators (GECIs) are suitable for long-term imaging studies. In this study, we employed a highly sensitive GECI, G-GECO, and achieved efficient gene delivery with an adenoviral vector. The adenoviral vector allowed us to express G-GECO in more than 80% of cells. More than 80% of G-GECO-expressing cells showed an ATP-induced increase in fluorescence intensity due to Ca²⁺ release from intracellular stores and subsequent Ca²⁺ entry. The fluorescence intensity of these cells was increased more than 2-fold by stimulation with 10 μM ATP. We applied long-term imaging (for ~10 h) to monitor Ca²⁺ responses in SF2, a rat dental epithelial cell line, in culture conditions. SF2 cells showed intermittent rises in the intracellular Ca²⁺ concentration in the presence of 100 nM 1,25-dihydroxyvitamin D₃. Many of these Ca²⁺ responses began at a specific location in the cytoplasm and spread throughout the entire cytoplasm. The combination of efficient gene delivery with an adenoviral vector and long-term imaging with a highly sensitive GECI enabled detection of intermittent Ca²⁺ responses that occur only 3–10 times/h/ 100 cells. This method could be useful to study the effects of Ca²⁺ responses for regulating longterm processes, such as gene expression, cell migration, and cell division, in many cell types.