Hoechst 33342 assay kit

The apoptosis of HeLa cells and HT-29 cells was detected using the Hoechst 33342 assay kit (Beyotime Institute of Biotechnology, China). HeLa cells (2 × 10⁵ cells/well) were seeded into a 6-well plate and treated with HES (0, 40, 80, and 160 μM) for 48 h. Then the attached cells were washed with phosphate buffered saline (PBS) and fixed with freshly prepared 4 % paraformaldehyde for 30 min. After fixation, the cells were washed with PBS and incubated with Hoechst 33342 staining solution for 5 min. After staining, cells were washed with PBS and anti-fade mounting medium (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China) was added, then the cells were viewed with a fluorescence microscope (Nikon Corporation, Tokyo, Japan). Apoptosis, as indicated by condensed and fragmented nuclei, was observed and recorded with the fluorescence microscope.
The HeLa cells (2 × 10⁵ cells/well) were seeded into a 6-well plate and treated with HES (0, 40, 80, and 160 μM) for 48 h. Then the attached cells were washed with PBS and the DNA was isolated from HES-treated and control cells use DNA isolation kit (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China), separated by 1.0 % agarose gel electrophoresis, viewed and photographed by an ultraviolet light gel documentation system.

Cell apoptosis of BV-2 cells was also detected using the Hoechst 33342 assay kit (Beyotime Institute of Biotechnology, China). In brief, BV-2 cells seeded on coverslips in a 6-well plate were pretreated with 5 μM Aβ(1-42) for 12 h, then followed with an incubation of 0.12–0.48 mg⋅L^-1 LSF for another 12 h. After treatment, BV-2 cells were fixed in freshly prepared 4% paraformaldehyde for 15 min and washed with PBS for three times, then incubated with 5 mg/L Hoechst 33342 staining solution for 5 min. After incubation, the air-dried slides were mounted with FluorSave^TM mounting media (Calbiochem, San Diego, CA, United States) and subjected to fluorescence microscopic analysis for observing the apoptosis under the microscope (Invitrogen EVOS FL Auto Cell Imaging System). The cells with condensed and fragmented nuclei were considered to be apoptosis.

HCT116 cell apoptosis induced by BSO was also assayed using the Hoechst 33342 assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Briefly, HCT116 cells were seeded into 6-well plates supplemented with sterile coverslips and treated with BSO (62.5, 125, and 250 μg/mL). After 36 h of culture, the medium was removed and the attached cells were carefully washed with PBS, and cells were then fixed with fresh ice-cold 4% paraformaldehyde (PFA) for 30 min at 4 °C. After fixing, cells were washed with cold PBS and incubated with Hoechst 33342 staining solution at a final concentration of 15 µg/mL for 10 min. The coverslips were subsequently washed with PBS and mounted using an anti-fade fluorescence mounting medium. Additionally, then, apoptotic cells with condensed and fragmented nuclei can be detected using fluorescence microscopy.