I’m using the Senescence β-Galactosidase Staining Kit from Cell Signalling to identify senescent cells. Does anyone know a method of reliably quantifying the stained cells in culture? I perform the assay in 12-well plates. Additionally, I’m not sure I have the pH correct due to the high amount of stained cells at baseline. Has anybody else experienced this issue?
Many thanks.
The assay has several requirements (see details in our paper in a link below)- the pH needs to be acidic (pH 6.0) and control cells need to have no or minimum background. If the assay is continued for a long-time, the number of positive cells increases in control cells. Therefore, the assay time (incubation time) needs to be optimized.
Link: http://www.nature.com/onc/journal/v22/n31/pdf/1206754a.pdf
Hi,
normally, primary cultures from early passages often consist of a fraction of cells that express SA-b-Gal. In fibroblasts this fraction accounts up to 10%, whereas in mesothelial cells even up to 50%. This is often called sudden senescence syndrome or simply premature senescence. This phenomenon is strongly associated with culture conditions, in particular with the method of cell isolation from a tissue (enzymatic digestion generates more senescent cells at first confluency than cell migration from an explant).
Good luck!
yes I got the same, even when I took well care of the ph before staining. It could be a result os premature senescence of culture related senescence.
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