Senescence-associated beta-galactosidase activity is a widely used biomarker for assessing replicative senescence in mammalian cells. This enzymatic activity has generally been measured by staining cells with the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal) at pH 6. 0, a reaction condition that suppresses lysosomal beta-galactosidase activity sufficiently to ensure that most nonsenescent cells will appear unstained.
coli JM109, DH5a and XL1-Blue strains).  In the presence of IPTG in growth medium, bacteria synthesise both fragments of the enzyme. Both the fragments can together hydrolyse X-gal (5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside.
Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) were obtained from BioWhittaker (Walkersville, MD, USA). Doxorubicin, 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl tetrazolium bromide (MTT), cisplatin, and 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-Gal), and protein A agarose were purchased from Sigma (St. Louis, MO, USA). Antibodies to p21/WAF1 and to -actin were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA), antibodies to phosphorylated p53, cleaved caspase-3 and secondary antibodies conjugated to horseradish peroxidase from Cell Signaling Technologies (Beverly, MA, USA).