*4.5. Observation of Morphologic Changes*

Cells were seeded in 24-well plates at a density of 1 × 10<sup>5</sup> for 24 h. After treatment for 24 h, cells were washed with phosphate-buffered saline and stained with 400 μL Hoechst 33,342 (2.5 μg/mL) for 5 min in the dark. After removing the medium and washed twice with phosphate buffer solution (pH 7.4), 400 μL PI (12.5 μg/mL) was added for 5 min in the dark. Cells with typical apoptotic nuclear morphology such as nuclear shrinkage and fragmentation and micronuclei formation were identified under fluorescent microscope and counted using randomly selected fields on numbered slides. The percentage of apoptotic cells was scored by counting at least 200 cells per treatment group and the average percentage of apoptotic cells was determined for each UF treatment and expressed as the mean ± SD.
