I'm working on apoptosis but recently found that instead of dying, my C17.2 cells were going into quiescence, (I think it is anyway, as it is reversible once the insult is removed). Just want to be sure what I'm looking at. Any good markers?
Id suggest some starting points:
Proliferation markers : Ki67, PCNA, Cyclin dependant kinases
Inflammation markers : NFKB, AGER
Apoptois: Caspases (intrinsic pathway vs extrinsic pathway)
Necrosis markers?
Consider all the four phenomenon and see which are most relevant to use as positive control or negative control. Then you can start hypothesising which way your cells are headed.
Read the article on this link : http://www.ncbi.nlm.nih.gov/pubmed/19833516
Also try this : http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030091
Hi Rajesh
Thanks for the post and the articles - really useful. I have ruled out apoptosis and necrosis using markers and inhibitors but will have a look at the others you mentioned. I'm also trying to differentiate between quiescence and senescence - I was wondering about p21 to discriminate?
Claire
Clue: Senescence is an outcome of Hayflick limit. Primary cells that have been passaged over and over are said to lose potential to replicate, this is a layman's idea of senescence. Please not viral transformation can counteract senecesence therefore immortalized lines should be avoided!
I work on senescence and unfortunately there is no specific marker that can directly detect Quiescent cells. You just need to check that they are not senescent using senescent markers and show the cells can re-enter the cell cycle if quiescent. Cells may also undergo a transient growth arrest to allow time to repair DNA damage and renter once repaired.
Thanks Dominick, that was also my impression. Do you consider your transient growth arrest as quiescence as well? How transient is transient? :)
Any luck with trying senescence induced b-galactosidase activity? It is a long shot but may give some clues... There is a kit available in the market. It works quite well on adherent cells.
I wouldn't class transient growth arrest as quiescence. "transient" I would say is within a 48hr period max, probably shorter. I have known some cancer cells to become senescent-like for several days before escaping the senescent program.
There are two primary things that you may want to consider to check for quiescence/senescence. Try performing a B-galactosidase staining with your cells. If you get that positive, you can confirm it as cell senescence. Thereafter, you can look at the expression of P16INK4A. That is a protein that is primarily responsible for cell senescence. Moreover, you can check for cell differentiation by conducting an Oil Red O staining. Hope this helps
@William, I did wonder about autophagy but I didn't see any differences when I look at LC3 processing by western blot.
@Neel, great, will try the B-gal assay.
@Dominick, my cells can stay "dormant" for 72h but I can reverse this with my protective compound from 24h post insult, so definitely veering towards quiescence
Have you look Ki67 expression ? This nuclear antigen is expressed in cycling cells. According the Ab, the staining is efficient in immunohisto (-fluorescence)-chemistry or flow cytometry. As mentioned p16 or B-galactosidase staining constitutes also a good test.
Hi Marie-Helene,
Yes I had a quick look by IHC at Ki67 on my cells and just from a rough counting, there were less in the insult and more in the insult + protectant which is what prompted me to think that it was maybe quiescence rather than senescence. But as I came across this completely by accident, I'm just checking to make sure that I haven't missed obvious markers. As you and @Neel both suggest, I should probably take a look at p16 as well.
Thanks
Claire
Yes,
P16 or B-galactosidase staining is more efficient to evaluate senescent cells (which are quiescent !).
SA-B-Gal is more a marker of lysosomal mass rather than specifically cellular senescence, so a combination of senescence markers will be needed to confirm senescence. Also, if cells are too confluence you can get artifactual staining for SA-B-Gal.
The accumulation of senescence-associated heterochromatin foci (SAHF) is another novel more specific biomarker of senescent cells，because there is marked focal formation of heterochromatin in the aged cells.
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