Quantitative assay of senescence-associated beta-galactosidase activity in mammalian cell extracts.
Anal Biochem
; 343(2): 329-34, 2005 Aug 15.
Article
in En
| MEDLINE
| ID: mdl-16004951
ABSTRACT
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. This article describes a quantitative method for measuring this activity and characterizes the method using extracts from senescent, quiescent, and presenescent human fibroblasts. The assay is capable of detecting relatively subtle changes in activity and confirms previous indications based on staining that confluency and contact inhibition of growth can cause a small increase in the expression of this biomarker. Investigation of the pH dependence of the activity in the cell extracts suggests that the senescent phenotype is correlated with an increase in total beta-galactosidase rather than with a shift in the pH optimum of the enzyme. This assay for measuring senescence-associated changes in beta-galactosidase is suitable for mechanistic studies of senescence regulation in which graduated changes in biomarker expression may be anticipated.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
Aging
/
Beta-Galactosidase
Type of study:
Risk_factors_studies
Limits:
Animals
/
Humans
Language:
En
Journal:
Anal Biochem
Year:
2005
Document type:
Article
Affiliation country:
Estados Unidos