S1060

Flow cytometric analysis was performed using a previously reported method [6 (link)]. For intracellular ROS measurement, the cells were treated with 5 μm H₂DCFDA (D399, Invitrogen) in PBS for 40 min at 37 °C in the dark, collected with 0.05% trypsin–EDTA, suspended in new media, and washed twice with PBS. To detect the intracellular calcium levels, the cells were stained for 30–40 min at 37 °C with the Ca²⁺‐sensitive fluorescent indicator Fluo‐4 AM (2 μm, S1060, Beyotime, Shanghai, China) or Fura‐2 AM (5 μm, S1052, Beyotime). A mitochondrial membrane potential assay kit with JC‐1 (C2006, Beyotime) was used to conduct the JC‐1 assay. Subsequently, flow cytometry analyses were performed using a BD FACS Canto II (BD Biosciences).

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.