Can be used to monitor and detect β-galactosidase activity in senescent cells at pH 6.0 using X-Gal.

Many primary cell types, particularly fibroblast cells, have a limited capacity to reproduce in cell culture. Even normal cells, derived from fetal, embryonic or newborn tissue, typically undergo between 40 and 60 cell divisions, but then often stop dividing, becoming senescent. When cells become senescent changes in morphology and gene expression patterns occur. In addition, senescence is also accompanied by increases in acidic senescence-associated β-galactosidase (SA-ß-Gal). This activity can be monitored using the substrate 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-Gal) at pH 6 and is a useful enzymatic marker of cellular senescence (SA-β-Gal). While cells entering senescence show this enzymatic activity, immortalized cell types, including tumor or other quiescent cells, are not stained under these conditions. The implications of these methods in such diverse areas as age-related pathology research, tumor analysis and tissue culture maintenance is significant. When β-Gal cleaves the glycosidic linkage in X-Gal, a soluble, colorless indoxyl derivative is produced, which quickly dimerizes and becomes oxidized to produce a bright blue indigo dye precipitate at the site of β-Gal activity. The dimerization and oxidation reaction is facilitated, in this kit, by a developer buffer solution containing ferric and ferrous ions. Senescent cells can be determined by examining the number of blue cells in the total cell population. The kit provides enough reagents and standards for up to 100 assays, using a microtiterplate method, and includes a detailed protocol for use in cultured mammalian cells.

This kit is sold by Marker Gene under product code M1389.