I have been trying to stain human lung fibroblasts isolated from lungs of young controls, aged and IPF patients for SA-Betagalactosidase expression. I need them to be in basal condition without any induction of senescence ideally. I have used the kit from Invitrogen that uses the traditional X-Gal stain on cells at roughly 75% confluence in a 12-well plate. I have followed the protocol that comes with the kit and it mirrors the protocol followed in studies that tested effects of induced senescence. Sadly, I could get only 5-6 cells to stain. I have the cells in passage 3-5. Could you suggest any modification of protocol or approach that you know has worked?
Hi, in my opinion you still have relatively young cells so the level of SA-b-Gal-positive cells in these populations is small. Your protocol is probably good and you can check this by inducing premature senescence in young fibroblasts e.g. by their treatment with hydrogen peroxide. If you'll get increased stainig, you'll get the proof that the method you use is correct. If you'll still have the positivity in only individual cells, there will be a rationale for some modifications of the protocol. Good luck.
I agree with suggestions by Krzysztof. Notably, cellular senescence is asynchronous in cultures. Therefore, if cells are young (which is likely), one does not expect all cells in culture to express senescence-associated beta-galactosidase. Please read the conditions for staining in the original PNAS paper (see the link below).
http://www.ncbi.nlm.nih.gov/pubmed/7568133
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