Measuring Intracellular Calcium Dynamics in INS-1D Cells

Intracellular calcium concentration ([Ca²⁺]i) in INS-1D cells was measured as previously described^{41 (link)} with a Calcium Fluo-4 kit (#CS22; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). INS-1D cells were placed on a φ35 mm glass bottom culture dish, the growth medium was removed and replaced with loading buffer containing 3 mM glucose and Fluo4-AM, and the culture dish was incubated at 37 °C for 1 h. Loading buffer was removed and replaced with recording buffer. [Ca²⁺]i levels were detected when stimulated with 25 mM glucose and 30 mM KCl. Fluo-4 fluorescent signal over the INS-1D cell area was detected by excitation at 488 nm on an LSM 510 Live microscope (Carl Zeiss, Oberkochen, Germany).