2.10. Detection of Apoptosis by Acridine Orange (AO) Propidium Iodide (PI) Double Staining

IMA- and S2TIN-induced cell death in MCF-7 breast cancer cells was quantified using AO and PI double-staining, according to standard procedures, examined under a fluorescence microscope. Briefly, treatment was carried out in T-flasks. Cells were plated at a concentration of 1 × 10⁶ cells/mL for 24 h, the media were substituted with new media containing 52.5 µM of IMA and 6 µM of S2TIN, along with a blank control. The cells were incubated for 72 h. Floating and adhering cells were collected, centrifuged at 300× g for 10 min, and washed twice with PBS to remove the remaining media. The supernatant was discarded, and the cells were stained with fluorescent dyes containing AO (10 mg/mL) and PI (10 mg/mL). The freshly stained cell suspension was dropped onto a glass slide and covered with a coverslip. The slides were observed under an ultraviolet fluorescence microscope within 30 min before the fluorescent color started to fade. The viable, early dependent phospholipid-binding protein, the plasma membrane alterations, and apoptotic and late apoptotic cells were observed.