What is the significance of plotting G2M/G0G1 ratios in cell cycle analysis.?
It is important to report all the cell cycle phases, in order to properly represent the entire cell population analyzed. If you made random searches for cell cycle analysis in the literature, you will see that all three cell cycle phases are reported in those papers.
The ratio will give an indication of dividing cells (cells in G2/M) to non-dividing cells.
As cells in G2/M will have 2x as much DNA as cells in G0/G1 then they can be distinguished using simple DNA stains such as DAPI) on a FACS.
As a rule of thumb around 10% of cells will be in G2/M at any time. If a growth factor is added to the cells (and they express a receptor for it) then the % may increase to 15-20% . Similarly if a DNA chelating agent (or similar) is added to the cells (which binds DNA and prevents cell division) then the % in G2/M may drop to
You could use this ratio (and some others, more exact) to estimate a specificity of action of different factors which can block cell division at definite "sensitive" stage of cell cycle.
Personally I would not use a G2M/G0 "ratio" as this does tend to ignore all those cells in S phase (if cells began accumulating at this check point then G0 and G2/M would begin to "empty" of cells but the ratios might not reflect this initially- although it would be pretty obvious on the FACS plot.).
I would just report the % of cells in each part of the cell cycle (ie: 80% G0/G1, 10% S, 10% G2/M). Ratios could then be applied between cells under different conditions if desired.
I agree with Dr. Morley. After knowing initial state in "control variant" different phase ratios could help evaluating the phase shifts of the cell cycle after exposure to various specific factors.
Im sorry to sound a little slow but I still don't understand the significance of the G2M/G1 ratio?
When graphing this ratio I should express it as a percentage?
I should also show the % of each phase when showing my data?
If the ratio is >10% what is the significance of this? does it mean that whatever I treated my cells with has pushed the cells into the G2 phase of the cell cycle?
Thanks in advance for your help and patience in answering what Im sure are silly questions!
Kind regards,
Eleanor
Hi Eleanor it is a good thing (rather than a bad thing) to ask questions.
You more or less have it.
The G2M:G1 ratio is a crude way of looking at how many cells are in the process of dividing in your culture.
For example I have some Jurkat cells growing normally and some Jurkat cells I have starved of FBS (which contains growth factors etc necessary for cells to survive in the long term). When I perform a cell cycle analysis on the FACS (which can just be done using a nuclear dye) in my control I may have 10000 cells in G2-M and 100000 in G1. The ratio here is 1:10 indicating 10% of my control cells are in the process of dividing at any one time. In my starved cells I may get 1000 "events" in G2-M and 200000 in G1. The ratio here is 1:200 indicating that only 0.5% of these cells are actively dividing. i.e. either something is stopping the cells in the starved condition dividing or something is no longer promoting cell division (the latter is most likely if the cells have been starved).
Without control cells the significance of the ratio of how may cells are in G2/M in the other condition is severely reduced (after all if you do not examine "normal" cells how can you interpret the cells in the starved condition (or any other condition such as in the presence of a dug or growth factor)).
To answer your second question: yes I would give the percentage of the cells present in all of the stages of the cell cycle that you can determine (i.e.: control cells: 70% G0/G1 20% S 10% G2/M cells - FBS: 95% G0/G1 2% S and 3% G2/M) as this adds more detail.
Whilst there are 3 major cell cycle check points (located at G1, G2 and M http://en.wikipedia.org/wiki/Cell_cycle_checkpoint) which should be covered by the G2M/G1 ratio it is not beyond the bounds of possibility that a drug etc. could affect the S phase and cause cells to accumulate here (which would be missed by the G2M/G1 ratio: eg: Drug "X" 7% G0/G1 92% S 1% G2/M. In this example the ratio of the G2M/G1 ratio is identical to the control mentioned above (7:1) but it is very clearly different with the vast majority of the cells accumulating in S phase.
I hope that makes sense?
Gary
Dear Dr Morley,
Thank you very much for replying to my question with such patience, it is much appreciated! Do you know if there is a reference that you could recommend that I could refer to regarding 10% of the cells being expected to be in the G2/M phase at any time. I have had a bit of a hunt around but cant find anything and would like to be able to reference this. Sorry to be troublesome and thank you again for your help.
Kind regards,
Eleanor
I'm afraid that is just from personal experience and is only an approximate value. The % of cells that are cycling at anyone time rather depends on the cell/type and it's environment. When I have time I will have a hunt around the web and see if anyone has published anything more precise although as mentioned it is situation dependent and so any info may be in regard to a specific case (i.e. people looking at the effect of cancer drugs on a cell).
Gary
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