Apoptosis analysis using Hoechst 33258 staining

Hoechst 33258 staining was used to investigate the nuclear morphologic changes of apoptotic cells [16]. Briefly, after exposure to different concentrations of L-securinine (2.5, 5, and 10 μM) for 48 h, cells were washed with cold PBS and fixed with freshly prepared 4% paraformaldehyde for 15 min at room temperature. Then the fixed cells was rinsed with PBS and stained with 500 μl Hoechst 33258 solutions (10 μg/ml) in compliance with the manufacturer’s instructions. Finally, the cells were washed thrice with PBS and Hoechst 33258-stained nuclei were observed using an inverted fluorescence microscope (Olympus, BX53, Japan).