Figure 1. Senescent astrocyte establishment and identification. (A–D) The astrocyte were treated with 20 g/L of D-galactose for different continuous passage culture time, and the senescent cells rate (bule staining cells) was detected using β-galactosidase staining; (E, F) The cells were treated with 20g/L of D-galactose for continuous passage culture of two generations, and the damaged DNA fragments was detected by single cell gel electrophoresis; (H–K) TUNEL (Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling) staining and DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) staining, DAPI stained all cells blue and TUNEL kits labeled apoptotic cells with green fluorescence; (L) The cell proliferation rate, (M) apoptosis rate and (N) cell cycle measurement after treated with 20 g/L of D-galactose for different continuous passage culture time; (O) Western blotting of P16INK4a and P21Cip1/Waf1 protein expressions; (P) The cell morphology with Giemsa staining at different time on the 20 g/L of D-galactose concentration. D0 (culture without 20 g/L D-galactose), D1 (culture in 20 g/L D-galactose for one generations) and D2 (culture in 20 g/L D-galactose for two generations) (Figure 2C, 2D), D3 (culture in 20 g/L D-galactose for three generations). Data are presented as the means±SD of 3 independent experiments. *p <0.05 and **p <0.01 vs. the control (normal or D0) group by one-way ANOVA, followed by the Holm-Sidak test.