A quantitative chemiluminescent method for studying replicative and stress-induced premature senescence in cell cultures

Abstract

β-Galactosidase (β-Gal) activity is a widely accepted biomarker to detect senescence both in situ and in vitro. A cytochemical assay based on production of a blue-dyed precipitate that results from the cleavage of the chromogenic substrate X-Gal is commonly used. Blue and nonblue cells are counted under the microscope and a semiquantitative percentage of senescent cells can be obtained. Here, we present a quantitative, fast, and easy to use chemiluminescent assay to detect senescence. The Galacton chemiluminescent method used to detect the prokaryotic β-Gal reporter enzyme in transfection studies was adapted to assay mammalian β-Gal. The assay showed linear production of luminescence in a time- and cell-number-dependent manner. The chemiluminescent assay showed significant correlation with the cytochemical assay in detecting replicative senescence (Pearson r = 0.8486, p < 0.005). Moreover, the chemiluminescent method (Galacton) also detected stress-induced senescence in cells treated with H 2O 2 similar to the cytochemical assay (X-Gal) (Galacton: control 25,207.3 ± 6548.6, H 2O 2 52,487.4 ± 16,284.9, p < 0.05; X-Gal: control 41.31 ± 7.0%, H 2O 2 92.97 ± 2.8%, p < 0.01). Thus, our method is well suited to the detection of replicative and stress-induced senescence in cell culture.