4.6. Propidium Iodide (PI)/Hoechst 33342 Double Staining

MCF-7 cells were treated with different concentrations of Rh1 for 24 h. After the treatment, the cells were washed with PBS before stained with PI (10 µM) at 37 °C for 10 min. The cells were then washed with PBS, stained with Hoechst 33342 (5 µg/mL) for 10 min, and fixed in 4% formaldehyde for 10 min. After being washed with PBS, the cells were mounted with Prolong Gold Antifade Reagent (#{"type":"entrez-protein","attrs":{"text":"P36934","term_id":"549428"}}P36934, Molecular Probes, Inc., Eugene, OR, USA). Subsequently, the images were taken, and a number of cells were quantified using a laser scanning confocal spectral microscope (K1-Fluo, Nanoscope systems, Daejeon, Korea), and a total of 6 fields per sample were counted. Hoechst 33342 (blue fluorescence) stained the nuclei of both live cells and dead cells, whereas PI (red fluorescence) only stained the nuclei of dead cells. Therefore, the cells stained with PI were indicated as dead cells.