Measurement of Mitochondrial and Cytoplasmic Calcium

Mitochondrial and cytoplasmic Ca²⁺ measurements were performed using Rhod-2 AM [31 (link)] (ab142780; Abcam) and Fluo-4 AM [32 (link)] (S1060; Beyotime). Cells were incubated with 5 µM dihydro Rhod-2 AM or 5 µM Fluo-4 on glass-bottomed dishes. The cells were then washed with HBSS and images were recorded using a Leica SP8 confocal microscope at excitation 549 nm/emission 578 nm or excitation 488 nm/emission 520 nm for green and red fluorescence, respectively. Subsequently, the cells were challenged with EBSS cultured in medium containing 10% FBS. Scans were collected every 15 s for 10 min under identical conditions. The fluorescence intensity at all the time points was processed using ImageJ software, and the region of interest (ROI) was selected.