Quantitative assay of senescence-associated β-galactosidase activity in mammalian cell extracts
Quantitative assay of senescence-associated β-galactosidase activity in mammalian cell extracts
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DOI：
10.1016/j.ab.2005.06.003
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发表时间：
2005-08-15
影响因子：
2.9
通讯作者：
Kindell, SM
中科院分区：
文献类型：
--
作者：
Gary, RK;Kindell, SM
衰老相关的 β-半乳糖苷酶活性是一种广泛使用的生物标志物，用于评估哺乳动物细胞的复制衰老。这种酶活性通常通过用显色底物 5-溴-4-氯-3-吲哚基-β-D-吡喃半乳糖侧 (X-gal) 在 pH 6.0 下对细胞进行染色来测量，该反应条件可充分抑制溶酶体 β-半乳糖苷酶活性，以确保大多数非衰老细胞不会被染色。本文描述了一种测量这种活性的定量方法，并描述了使用衰老、静止和衰老前人类成纤维细胞提取物的方法。该测定能够检测相对细微的活性变化，并根据染色确认先前的指示，即生长的汇合和接触抑制可能导致该生物标志物表达的小幅增加。对细胞提取物中活性的 pH 依赖性的研究表明，衰老表型与总 β-半乳糖苷酶的增加相关，而不是与酶的最适 pH 值的变化相关。这种用于测量β-半乳糖苷酶衰老相关变化的测定法适用于衰老调节的机制研究，其中可以预期生物标志物表达的逐渐变化。 (C) 2005 Elsevier Inc. 保留所有权利。
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-galactopyrano side (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. (C) 2005 Elsevier Inc. All rights reserved.