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

Autor(es)

Bassaneze‚ V. ; Miyakawa‚ A.A. ; Krieger‚ J.E.

• Tipo de documento: Artigo Científico
• Fonte: Analytical Biochemistry; v. 372, n. 2, p. 198-203, 2008.

Resumo

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. H2O, 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. (C) 2007 Elsevier Inc. All rights reserved. (AU)

• Processo FAPESP: 00/09485-7 - Identificacao de genes com expressao diferenciada em veia safena submetida ao regime arterial.
• Beneficiário: Ayumi Aurea Miyakawa Yamaguchi
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 05/57591-4 - Caracterizacao da influencia do shear stress na diferenciacao de celulas mesenquimais de tecido adiposo em celulas endoteliais.
• Beneficiário: Vinícius Bassaneze
• Modalidade de apoio: Bolsas no Brasil - Mestrado
• Processo FAPESP: 01/00009-0 - Estudo integrado da hipertensão arterial: caracterização molecular e funcional do sistema cardiovascular
• Beneficiário: Eduardo Moacyr Krieger
• Modalidade de apoio: Auxílio à Pesquisa - Temático