Senescence associated β galactosidase staining kit

For SA-β-galactosidase staining, cells were washed with PBS, fixed with 4% paraformaldehyde in PBS at room temperature for 20 min and incubated with reagents from a senescence-associated β-galactosidase staining kit (Beyotime Institute of Biotechnology, #C0602) according to the manufacturer’s suggestions.
For alkaline phosphatase staining, differentiated osteoblasts were washed with PBS, fixed with 4% paraformaldehyde for 30 min at room temperature and stained with an Alkaline Phosphatase Staining Kit (Beyotime Institute of Biotechnology) for 1 h at room temperature in the dark.
For alizarin red staining, cells were fixed with paraformaldehyde for 30 min, incubated with 1% alizarin red for 30 min at room temperature and washed with PBS to remove the excess dye.
For immunocytochemical staining, we incubated cultured cells with primary antibodies recognizing mouse Ca_v1.2 (Abbkine, 1:100, #Abp57305) or NFATc1 (Santa Cruz Biotechnology, 1:100, #sc-7294) overnight at 4 °C. Secondary antibodies conjugated with fluorescent tags were incubated at room temperature for 1 h in the dark.

For SA-β-galactosidase staining, cells were washed with PBS, fixed with 4% paraformaldehyde in PBS at room temperature for 20 min and incubated with reagents from a senescence-associated β-galactosidase staining kit (Beyotime Institute of Biotechnology, Shanghai, China, #C0602) according to the manufacturer’s suggestions.
For alkaline phosphatase staining: Treated C3H10T1/2 cells were washed with a phosphate buffer, fixed in 4% paraformaldehyde for 30 min at room temperature, and stained with an alkaline phosphatase staining kit (Beyotime Institute of Biotech, Shanghai, China) for 1 h at room temperature in the dark.
For alizarin red staining, cells were fixed with paraformaldehyde for 30 min, incubated with 1% alizarin red for 30 min at room temperature and washed with PBS to remove the excess dye.

Fresh tissue was embedded with a frozen section embedding agent and sliced at −20 °C. SA-β-gal staining was performed using the senescence-associated β-galactosidase staining kit (C0602, Beyotime Co., Shanghai, China). Oil Red O staining was performed using Oil Red O (OILR-10001, Cyagen Biosciences, Guangzhou, China). After staining, the nuclei were re-stained and then sealed for observation.

For senescence-associated β-galactosidase (SA-β-gal) staining, dCLN CD8⁺ T cells were isolated, fixed, and stained for SA-β-gal at 37℃ overnight by Senescence-associated β-Galactosidase Staining Kit (Beyotime, Cat# C0602) according to the manufacturer’s instruction. For quantification of SA-β-gal-positive cells, images were randomly taken at ×40 magnification (BX-63, Olympus) and then analyzed manually with ImageJ.

To measure the senescence activity in the GCs, we used the Senescence-associated β-Galactosidase Staining kit (Beyotime, C0602, China) following the manufacturer’s instructions. Briefly, seeded GCs in a 6-well plate in an appropriate density. Then it was treated with M0 supernatant, M1 supernatant, Metformin, etc. in different groups. When the processing time was reached, GCs were fixed with 4% paraformaldehyde for 15 min and then washed with PBS 3 times. Eventually, add β-Gal substrate and according to the instructions incubate overnight at 37 °C. Subsequently, after three washings with PBS, the positive senescent cells appeared blue-green with a light microscope. The degree of senescence was proportional to the number of blue-green cells.