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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 Ca 2+ release from intracellular stores and subsequent Ca 2+ 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 Ca 2+ responses in SF2, a rat dental epithelial cell line, in culture conditions. SF2 cells showed intermittent rises in the intracellular Ca 2+ concentration in the presence of 100 nM 1,25-dihydroxyvitamin D 3 . Many of these Ca 2+ 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 Ca 2+ responses that occur only 3-10 times/h/100 cells. This method could be useful to study the effects of Ca 2+ responses for regulating longterm processes, such as gene expression, cell migration, and cell division, in many cell types.

biomedical research

&lt;b&gt;Effect of 1,25-dihydroxyvitamin D&lt;/b&gt;&lt;b&gt;&lt;sub&gt;3&lt;/sub&gt;&lt;/b&gt;&lt;b&gt; on spontaneous calcium responses in rat dental epithelial SF2 cells revealed by long-term &lt;/b&gt;&lt;b&gt;imaging &lt;/b&gt;