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-ÃŸ-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.
References and Citations
Dimri GP, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE,
Linskens M, Rubeli I, Pereira-Smith O, Peacocke M, Campisi J (1995) "A biomarker that identifies senescent human cells in culture and in
aging skin in vivo." PNAS 92: 9363-9367.
Nakamura M, Kondo H, Shimada Y, Waheed AA, Ohno-Iwashita Y. (2003) "Cellular aging-dependent on decrease in cholesterol in
membrane microdomains of human diploid fibroblasts." Exp. Cell Res.
Aoshiba K, Tsuji T, Nagai A. (2003) "Bleomycin induces cellular senescence in alveolar epithelial cells." Eur. Respiratory J. 22(3): 436-443.
We pride ourselves on the high quality of our products and want you to get the best possible results from your assays. If you have any questions about this product or need help optimizing your protocol check out the product FAQs below or ask your own question and one of our expert scientists will get back to you asap:
⚛ Question: I am working with the Cell Senescence kit but I am not sure what to use as a
control. Can you suggest either a negative or a positive control (or
preferably) both a negative and a positive control.
➸ Answer: For a positive control please see the following references for suggested methods of inducing senescence in cancer cell lines:
Roninson IB. Tumor cell senescence in cancer treatment. Cancer Res. 2003 Jun
For a negative control a culture of low passage number of the experimental cell line should not exhibit any senescence markers.
⚛ Question: Can I use this kit for stratified cells?
➸ Answer: We have not tested the use of this kit in stratified cells however several customers have successfully used the kit for staining of tissue sections which would suggest that the dye is capable of penetrating several layers of cells.
⚛ Question: I am researching a chemical that is supposed to inhibit cellular senescence. What would be the best way to add the chemical to the cells in order to measure the senescence?
➸ Answer: In order to measure the effect of a chemical on your cells I would suggest that you plate your cells in media containing the chemical at an appropriate concentration and then incubate for an appropriate amount of time prior to performing the senescence assay.
⚛ Question: Can cells be fixed and then stained the following day?
➸ Answer: You can try fixing the previous day ensuring that you rinsing with PBS several times to remove the excess fixative solution, but nevertheless, leaving the cells overnight or for several nights may reduce the levels of b-Gal activity remaining in the cells, even if they are stored at 4oC.
It is recommended that the staining be performed immediately or soon after the PBS rinse to see optimum results. Remember that a hallmark of the acidic senescence-associated ?-galactosidase (SA-b-Gal) activity is that the assay is performed at low pH as it has a pH optimum of about 6.0.