Apoptotic aate of cells by flow cytometry assay and Hoechst 33342 staining

The apoptotic rate was measured by flow cytometry as described previously [7, 8]. Cells were washed three times with ice-cold PBS, and then stained with annexin V-fluorescein isothiocyanate for 15 min at room temperature in 200 μl binding buffer. Next, 300 μl binding buffer was added, and the cells were stained with propidium iodide for 30 min at 4 °C. The fluorescence of the cells was analyzed by flow cytometry. The percentage of apoptotic cells was determined using Mod Fit LT software (Verity Software House Inc., Topsham, ME, USA).

Cells were analyzed for apoptosis after visualization of nuclei morphology with fluorescent DNA-binding dye Hoechst 33342, as described previously [8]. After treatment, cells were rinsed with PBS and incubated with 5 μg/ml Hoechst 33342 for 10 min. Nuclei were visualized at 400× magnification using fluorescent microscopy at an excitation wavelength of 330–380 nm. Apoptotic nuclei of cells were assessed by counting the number of cells that displayed nuclear morphology changes, such as chromatin condensation and fragmentation.