Intracellular Calcium Concentration Measurement

The concentration of intracellular Ca²⁺ was determined using a cell-permeable fluorescent calcium indicator, Fluo 4-AM. Cells were treated with DMEM-F12 and 10% bovine serum for 24 h and washed 3 times with D-Hanks balanced salt solution without Ca²⁺ (Ca²⁺-free HBSS). Subsequently, cells were loaded with 2μmol/l Fluo 4-AM for 30 minutes at 37 °C in the dark, then washed 3 times with Ca²⁺-free HBSS to remove the extracellular Fluo 4-AM. The solution was replaced with HBSS with Ca²⁺ before testing. Imaging was performed using the Leica SP8 Confocal Inverted Microscopy (Leica, Mannheim, Germany) and analyzed with Image-Pro Plus 6.0. We used the measured average fluorescence intensity of each cell in the field (F), normalized to the non-specific background fluorescence (F0) to obtain the fluorescence intensity (F/F0)^{22 (link)}.