Hoechst 33258 staining was performed as described previously with minor modifications . Briefly, cells were fixed with 4% formaldehyde solution for 30 min at room temperature and washed twice with PBS. Fixed cells were stained with Hoechst 33258 of 50 ng/mL and incubated for 30 min at room temperature and washed with PBS. Apoptotic cells were identified by condensation and fragmentation of nuclei examined by an Olympus IX71 fluorescence microscope. The apoptotic rate of cell population was calculated as the ratio of apoptotic cells to total cells counted ×100. A minimum of 500 cells were counted for each treatment.