4.1.6. Measurement of Mitochondrial Calcium

Mitochondrial calcium was measured using rhod-2/AM (TMRE; Thermo Fisher Scientific). Cells were incubated with 1 μM rhod-2 for 30 min. After PBS washing, the fluorescence was measured at 552 nm excitation/581 nm emission and cells were imaged using a fluorescence microscope.

#### 4.1.7. Cell Migration Assay

Scratch wound assays were used for cancer cell mobility analyses. MCF-7 and HeLa cells were seeded into 25-well cell culture plates at a concentration of 2 × 104 cells and were maintained in 10% FBS-DMEM until 70–80% confluent. The scratch was carefully made using a 20P sterile pipette tip. The remaining cellular debris was gently removed with PBS. The wounded monolayer was incubated in 10% FBS-DMEM containing 10 μM SpD for 24 h. The cell migration was observed under 4× by using a phase contrast microscope.
