Rhod2 am

The detection of calcium distribution was visualized by staining with Fluo4-AM (37 °C for 30 min; Dojindo, Japan) and Rhod2-AM (4 °C for 30 min; Dojindo, Japan) for the intracellular and intramitochondrial calcium, respectively. The nuclei were visualized by staining with Hoechst 33342 (Dojindo, Japan).

Changes in the cytosolic Ca²⁺ ([Ca²⁺]_cyt) and [Ca²⁺]_mit levels were measured using Fluo 4-AM and rhod 2-AM (Dojindo Laboratories), respectively as previously described (46 (link),47 (link)). For the improvement of mitochondrial localization of rhod 2-AM, it was reduced to the colorless, non-fluorescent dihydrorhod 2-AM by sodium borohydride, according to the manufacturer’s instructions. The cells were loaded with 4 µM each of Fluo 4-AM or dihydrorhod 2-AM for 40 min at 37°C, washed with HBSS. Subsequently, the cells (1×10⁶/ml) were resuspended in HBSS in 96-well plates. The cells were then manually supplemented with the agents to be tested PSM (12.5–50%), 100 ng/ml TRAIL, 5 mM KCl, 100 µM glibenclamide, 100 µM U37883A alone or in combination for 1, 3, 5 and 10 min. The cells were then measured for fluorescence using a microplate reader (Fluoroskan Ascent) with excitation and emission at 485 and 538 nm (for Fluo 4-AM) and 542 and 592 nm (for rhod 2-AM), respectively. For the analyses of Ca²⁺ release and store-operated Ca²⁺ entry (SOCE), Fluo4-AM-loaded cells were suspended in a Ca²⁺-free medium (HBSS supplemented with 1 mM CaCl₂) and treated with 1 µM thapsigargin (Tg) for 10 min and then added with 2 mM CaCl₂. The fluorescence was measured as described above with excitation and emission at 485 and 538 nm.