Quantifying Premature Senescence in H2O2-Treated Fibroblasts

Premature senescence was tested in fibroblasts treated with different concentrations of H₂O₂ for 1.5 h using senescence-associated (beta)-galactosidase (SA-β-gal) as a biomarker of senescence. After H₂O₂ treatment, the medium was replaced with fresh complete medium, and cells were cultured for 72 h before staining for SA-β-Gal activity as previously described [26 (link)]. Quantification of premature senescence was determined by calculating the rate of conversion of 4-methylumbellliferyl-β-D-galactopyranoside (MUG) to the fluorescent product, 4-methylumbelliferone (4-MU), at pH 6.0 as previously described [30 (link)]. The relative increase in 4-MU fluorescence per mg protein was determined by subtracting the untreated control values from the H₂O₂-treated values.