**5.2 Detection of morphological features of apoptotic cells by acridine orangeethidium bromide dual staining**

The morphological features of apoptosis induced by the compounds will be evaluated by acridine orange-ethidium bromide dual (AO/EtBr) staining [23]. Briefly, cells

**Figure 4.** *Apoptotic cells can be seen as fragmented DNA in smear appearance.*

#### **Figure 5.**

*Acridine orange-ethidium bromide dual staining: (a) and (b) apoptotic cells in orange color, live cells in green color.*

**Figure 6.** *Hoechst 33342: (a) non-apoptotic cells in blue color, (b) apoptosis cells in fluorescent blue color.*

will be seeded in a 96 well plate at a density of 5 × 105 cells and treat with different concentrations of compound for 24 h. After washing once with PBS, the cells can be stained with 100 μl of a mixture (1:1) of acridine orange-ethidium bromide (4 μg/ ml) solutions. The cells can be immediately washed with PBS and observed under fluorescence microscope at 450–490 nm. The effects of the compound treated on human oral cancer cells (KB) for 24 h can be visualize using fluorescent microscope (**Figure 5**).
