I´m trying to stain senescent cells along with the apoptotic ones and for some reason I cannot stain for Caspase 3 on top of X-gal staining. This is why I was suggested that it would be a good idea to try anti B-gal staining instead.
X-gal reaction is sensitive to B-galactosidase activity, while antibodies show the raw amount of B-galactosidase in the cells. Are these things equivalent in terms of detecting senescence, or it´s important to detect the activity exclusively?
I have looked into this a bit. It seems that people view X-gal staining as the gold standard for senescence, but I don't find papers in which the expression of B-galactosidase, either protein or mRNA, is measured. It's actually enzyme activity at low pH that indicates senescence, so perhaps the activity of the enzyme is modulated rather than the level (pure speculation, but maybe someone else knows). There are fluorescent assays for senescence associated B-gal if that helps. In addition, there are other markers that are considered to correlate with senescence in different cell types such as changes in laminB or upregulation of certain cytokines. Good luck!
Beta-galactosidase activity is a late response of the senescent cells that can be detected via X-gal staining. However, senescent cells usually start to express p21 earlier than beta galactosidase so this can also be checked. For the combination of the chemical staining of caspase-3 and Xgal, I do not know your protocol but one can block the cells then do the caspase 3 antibody incubation before fixation, then continue with Xgal staining.
I agree it is the activity of beta-galactosidase what you are looking for and, as Sila mentioned, p21 expression would further support your findings.
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