To Whom It May Concern,One question for the Beta-gal assay:...

Sep 5, 2012
protocol Protocol: Senescence Associated β-galactosidase Staining
To Whom It May Concern,

One question for the Beta-gal assay: will the stained cell number change (/increase) along with the incubation time? My experience is that I got ~40% blue cells at the 6th hour, and obtain ~90% blue cells at the 24th hour (they were in the same cell pool,
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chao fan Answered Jul 10, 2019

重庆市大坪医院心内科

Dear Bill, It's real surprising and even exciting to see your writing here,I‘m doing the same experiment with HUVEC,it's real hard to get the perfect result because all the cells became blue after 24h staining ,maybe I should shorten the time as you advised. Thanks very much!May I got your email?I hope to ask more question from you .
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Michael Eccles Author Answered Sep 5, 2012

Department of Pathology, University of Otago, New Zealand

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in our experience, different cell lines require different incubation times. If HUVEC naturally have high endogenous galactosidase, the pH of the incubation buffer will be critical. If you see blue cells ONLY in the treatment known to induce senescence, then 6 hours might be better, assuming this time point is in the linear range of the senescence development. Having a positive control will be important (e.g. using very late passage cells that have stopped dividing).

hope this helps.
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