4.7. Hoechst 33342 Staining and FACS Assay with Annexin V/PI Staining

We assessed apoptosis using Hoechst 33342 and fluorescence activated cell sorting (FACS) analysis. In Hoechst 33342 staining, PC-12 cells were seeded into six-well plates, after various treatment cells were stained with 1 μg/mL Hoechst 33342 for 5 min. The cells were washed and fixed with 4% paraformaldehyde in PBS for 5 min at 4 °C, then observed by fluorescence microscopy. Cells with condensed nuclei were considered apoptotic, and the percentage of apoptotic cells in PC-12 was determined by examining at least 300 cells per group, at ×400 magnification. The apoptosis assays were also conducted using an Annexin-V-FITC apoptosis detection kit by flow cytometry, according to the manufacturer’s instructions. PC-12 cells were exposed to various conditions treatment, and were harvested and washed twice with cold PBS, resuspended in 1× binding buffer. After incubation with Annexin V for 15 min in the dark at RT, the cells were immediately analyzed by flow cytometry.

TUNEL assay: PC-12 cells were seeded on coverslips of six-well plates for 24 h incubation, and treated with the indicated conditions. TUNEL assay was performed according to the manufacturer’s instructions to assessed apoptosis of various groups. Apoptotic cells were observed by a fluorescent microscope (BX51, OLYMPUS, Tokyo, Japan) at an excitation wavelength of 515–565 nm.

After the indicated treatment, organotypic hippocampal slice cultures were fixed with 4% paraformaldehyde in PBS for 30 min. TUNEL assay was performed according to the manufacturer’s instructions. At least five randomly-chosen areas in every slide were used. Percent apoptosis was determined by counting the number of apoptotic cells and dividing by the total number of cells in the areas.