2.11. Hoechst 33258 Staining

Hoechst staining was used to detect cell apoptosis. As cells undergo apoptosis, chromatin sequestrates. After Hoechst staining, the nuclei of normal cells appear normal blue, while the nuclei of apoptotic cells are abnormally bright or whitish in color. The transfected cells were seeded at 1 × 10⁵ cells/mL (3 mL per well) into 6-well plates. After the cells were incubated at 37°C with 5% CO₂ for 24 hours, the media was removed. The cells were then fixed with paraformaldehyde, washed with PBS, and stained with Hoechst 33258 (HY-15558, MedChemExpress, NJ, USA) in the dark. After 30 minutes, the cells were observed under a fluorescence microscope, and 5 fields were randomly chosen from each slide for photograph. The apoptosis rate was calculated according to the formula: apoptosis rate = number of apoptotic cells/total number of cells × 100%.