A quantitative chemiluminescent method for studying replicative and stress-induced premature senescence in cell cultures.
beta-Galactosidase (beta-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 beta-Gal reporter enzyme in transfection studies was adapted to assay mammalian beta-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 H2O2 similar to the cytochemical assay (X-Gal) (Galacton: control 25,207.3+/-6548.6, H2O2 52,487.4+/-16,284.9, p<0.05; X-Gal: control 41.31+/-7.0%, H2O2 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.
Bassaneze
Vinícius
V
Heart Institute (InCor), University of São Paulo Medical School, Av. Dr. Enéas de Carvalho Aguiar, 44-10 andar, 05403-000 São Paulo, SP, Brazil.
Miyakawa
Ayumi A
AA
Krieger
Jose E
JE
D015415:Biomarkers; D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division / Q000502:physiology*; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology*; D001026:Coronary Artery Bypass; D006801:Humans; D049449:Luminescence; D011994:Recombinant Proteins / Q000378:metabolism; D012501:Saphenous Vein / Q000166:cytology / Q000473:pathology / Q000502:physiology*; D013312:Stress, Physiological; D001616:beta-Galactosidase / Q000378:metabolism*
D015415:Biomarkers; D011994:Recombinant Proteins; D001616:beta-Galactosidase