**2.4 Apoptotic Index**

Apoptotic cells showed characteristic fragmented nuclei, whereas survivors showed an intact nuclear morphology. These cells were fixed with cold methanol. The cells were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) nuclear staining mounting solution for the determination of the apoptotic index (AI) [33]. Then, the slides were washed in phosphate-buffered saline (PBS). The AI was calculated by scoring the percentage of apoptotic cells in control and the extract treatment (0.1 mg/ml) group of 1000 cells and at least 30 areas/each slide under the fluorescence microscope [34].

### **2.5 Phase-contrast microscopies**

After the treatment with C. baytopiorum extract (0.1mg/ml), the morphological changes in HeLa cells were examined after 24, 48, and 72 h under phase contrast microscopies (x200).
