Senescence analysis using the Senescence β-Galactosidase Staining Kit

HCT116 (p53^+/+, p53^−/−, and p21^−/−) cells were plated in 6-well plate at a density of 10⁵ cells/mL. Cells were treated with 20 or 40 µg/mL GT and incubated for 72 hours at 50% confluency. At 72 hours, the growth medium was removed from the cells and the wells were washed with 2 mL 1 × PBS. The cells in each well were then fixed with 1 mL 1 × fixative solution provided by the Senescence β-Galactosidase Staining Kit (Sigma-Aldrich) for 15 minutes at room temperature. Cells were then washed twice with 2 mL 1 × PBS. To each well, 1 mL staining solution mix containing 930 µL staining solution, 10 µL staining supplement A, 10 µL staining supplement B, and 50 µL 20 mg/mL X-gal dissolved in dimethylphenol was added. The cells were incubated overnight at 37°C. The next day, the development of blue color was detected under the microscope at 100 × magnification. For quantification of senescent cells, 5 random regions were taken for each well and the percentage of senescing cells was calculated for each image.