2.5. Apoptosis Detection by Hoechst 33258 Staining

Hoechst 33258 staining was utilized to visualize nuclear changes and apoptotic body after treatment with selected compounds and MCEO. The cultures were treated with control, compounds, and/or MCEO and incubated during 48 h to analyze cell morphology. The cells were fixed in phosphate-buffered saline at 4°C for 30 min with 4% p-formaldehyde. Afterwards, the cells were washed in PBS, and nuclear DNAs were incubated with Hoechst 33258 fluorescent dye (1 M) at room temperature for 5 min. The morphological changes of cell nuclei were observed and photographed under fluorescence microscopy (Leica® DM IL LED).