Apoptosis Analysis of HeLa Cells

HeLa cells, seeded at 6 × 10⁴ per well in 12-well plates, were treated with test compounds for 48 h. Then, cells were collected and washed with PBS to a density of 5 × 10⁵ cells/mL. Hoechst 33,342 (10 ug/mL) was added to the cell suspension and incubated at 37 °C for 10 min. Thereafter, cells were centrifuged, resuspended in PBS, and PI (1 μg/mL) was added. After 15 min of incubation in the dark at room temperature, cells were placed on a u-slide 8-well chamber (Ibidi GmbH, Gräfelfing, Germany). The slides were immediately observed using an LSM 710 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany) under 40× magnification and analyzed with the Zeiss LSM 710 laser scanning confocal microscope (Carl Zeiss SMT, Inc., Oberkochen, Germany). The percentage of apoptotic cells was estimated by counting the number of chromatin condensed-positive nuclei and Hoechst-stained nuclei in six randomly selected 40× magnification images. The ratio between the number of chromatin condensed-positive nuclei and the total number of intact nuclei was calculated [24 (link),25 (link)].