β galactosidase

The senescent cells were identified with a β‐galactosidase Staining Kit (Beyotime), which uses X‐gal as the substrate and produces dark blue products under the catalysis of aging‐specific β‐galactosidase. Blue cells expressing β‐galactosidase can easily be observed under a light microscope. The cells were stained with β‐galactosidase, fixed at room temperature for 10 min and washed with 0.01 M PBS (Beyotime) three times for 5 min each. Then, a working dye solution comprising a mixture of β‐galactosidase staining solution A (10 μl), β‐galactosidase staining solution B (10 μl), β‐galactosidase staining solution C (930 μl), and X‐gal (50 μl) was added, and the mixture was incubated at 37°C for 4 h. The samples were observed, photographed and counted under an ordinary optical microscope. If photos were not taken sufficiently quickly, the dye solution was discarded, an equivalent volume of PBS was added, and the cells were stored at 4°C for several days. The proportion of positive cells was determined as the number of β‐galactosidase positive cells in 10 fields divided by the total number of cells and was compared through statistical analysis between groups.