I want to identify senescent cells in culture and have been using the B-Gal staining kit (Cell Signalling) with inconclusive results. I would like to confirm my observations using another method.
Any other ideas apart from WB?
Very grateful for any advice.
You can use other techniques where the antigen is in native conditions like ELISA and Dot Blot, likewise check the conditions in which you make your WB in relation to the denaturing and / or reducing conditions; So a WB but in native conditions could give you a better result.
I use a cell signaling kit to label b-gal+ senescent cells in culture during replicative lifespan experiments, as their morphology changes and they reach the end of their replicative capacity they will stain + (blue) if they are senescent. Maybe we are talking about the same kit (#9860)? I have never had problems and it doesn't get any easier. Some words of advice - you may have crystal formation and this can impede results or affect image quality so make sure all your reagents are properly thawed as per instructions and that the stains are in solution completely (vortex well and give some time to dissolve). The signal is pH sensitive so that may be your problem. This protocol also calls for dry incubation over night, sometimes I have had to incubate for over 24 hours to get good results and I have even incubated "wet" (normal conditions for live human cells) and that usually worked better, strangely. What cell line are you working with? I am not sure that SA-B-gal is a universal marker of senescence as there are different senescent programs and it is highly context dependent.
You can do qPCR and/or WB for other senescent markers such as hTERT, p16INK4a, p21, nuclear lamina proteins. This is common in the field. I have also used nuclear blebbing as a visual marker of senescence, it is an easy qualitative assessment if you are already doing IHC or other cell microscopy, just use any nuclear DNA fluorescent marker (I use hoescht and it works great) and you will clearly see nuclear morphology, some have even used this as a quantitative measure (see ref#1). You could also do ICC with an anti-lamin A (or lamin B?) ab and there are methods to detect nuclear foci that result from changes in chromatin that are associated with senescence.
1.http://www.aging-us.com/article/5WSHi52txe7hdALih/text
2.http://www.nature.com/nprot/journal/v4/n12/full/nprot.2009.191.html
Good luck,
Brenda
Hi Brenda,
Many thanks for your detailed answer.
I have indeed been using the same B-Gal staining kit but have been getting a large amount of staining at baseline (baseline: cells not induced into senescence or, at an early passage).
I have been using primary human lung fibroblasts and have been observing little/ if any discrimination in staining between experimental conditions.
As you mentioned, following the protocol is crucial especially achieving the correct pH. I have been in contact with the CS rep to confirm my methodology is satisfactory.
What type of cells have you been using when confirming senescence? And, do you get any staining at all at baseline?
My concern is that as B-gal is an enzyme used for breaking down B-galactosides, such as lactose, and that it should be present in all cells even at baseline. So, this makes validating senescent cells based on staining a matter of staining intensity which makes interpretation subjective.
Unfortunately, I’ve had a great deal of trouble in getting p21, and p16 to work successfully with WB. I haven’t yet tried hTERT. Thanks for your suggestions regarding IHC and ICC.
Best,
David.
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