Recently I checked the beta-gal staining status of my cells and found it significantly higher than in control cells. However, the cell cycle analysis using flow cytometer suggest there were no significant differences of cell cycle distribution (Actually, the cell cycle profiles of the two cell lines resemble each other, with 65% in G1, 25% in S and 10% in G2). Do you have any suggestion with this? Do I have arrest-and-release the cells before I do the cell cycle analysis?
The short answer is No. Fundamentally, senescent cells are permanently growth arrested. The problem is with the SA-b-Gal, as on its own it is not a reliable marker for senescence. For example, I believe macrophages can show positive staining for SA-b-Gal and their not senescent cells. Also, confluent cells can also demonstrate positive staining for SA-b-Gal. You will need to use other markers, such as p16, Ki67 (growth arrest), H2AX (DNA damage) to assess senescence.
Dear Dominick, Thanks for your thoughts. Actually we had successfully induce cell senescence(SA-beta-gal, proliferation assay, etc.) under our conditions, but in another cell line, accompanied by G2-M cell cycle arrest. Then we tested if the same treatment condition would induce cell senescence and cell cycle arrest. The SA-beta-gal was good and cells were obvious flattened and enlarged after the treatment, but we did not detect any differences in cell cycle distribution.
Anyway, thanks your advice.
Cell cycle profiles by FACS do not measure G1, S or G2/M but DNA content (2C, 4C etc.). You will need to perform BrdU (EdU) incorporation assays (or similar) to score lack of proliferation..
I agree with the views of Dominick. Senescence usually represents the growth arrested state of cells. So when SA-b-Gal assay is not very helpful, one can measure measure the levels of other markers like Ki67 and/or H2AX.
This is a very interesting question that relates a little bit to a question I posed: What is the G0 phase of the cell cycle, does it exist and how does it relate to a term such as "quiescence" and in your case "senescence". How clean is the definition of senescence nowadays? In keratinocytes it is morphologically obvious and results in a very bad growth arrest with little option to escape this growth arrest, almost or completely irreversible. Theoretically, most of us would associate this with the arbitrary term G0 phase of the cell cycle without giving it much thought. But my question is: does G0 really exist, is there a molecular marker?
If you do your experiment in vitro, how much does this relate to an in vivo senescence program. Most stuff we do in vitro is a little bit artificial. In vitro experiments are based on our need to have many cells and promote growth. Then it comes to studying senescence, we want to still have many cells but stop growth in an almost brutal fashion. Don't tell me this is not artificial, or isn't it? We learned so much from cell culture but in the end it is an approximation, a model, a simplification. In our minds however, we declare it realistic, truthful, representative, and equal to the "real" world. So we come up with fancy textbook terms such as G0 phase, senescence, quiescence. They are either very descriptive (senescence, quiescence) or highly artificial and almost meaningless (G0 phase).
But this is just my opinion, my reading of the world and I would have like to know from the forum whether there is more substance to these terms than I personally think there is. In the end there is a lot of inductive reasoning and not many brutally hard facts?
You could design an in vivo experiment, by comparing markers for the cell cycle/proliferation as suggested here using BrdU and a marker for senescence, try to co-stain and see how this all stacks up in a real animal, in the real world. Does that make sense?
The assay for replicative senescence is acceptable. However, additional criteria are needed to determine whether cells are permanently growth arrested. The assay, depending upon it is performed, could give a positive result. The original reagents along with the method need to be used for the assay with some caution.
Hello. I am in your same condition. I'd like to know what is your opinion now.
Thank you
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