increased s phase in teh cell cycle should also be complemented with an increased g2m phase?
in context to cell cycle analysis using PI ?
It depends on your experiment. For instance, say you had your cells treated with a drug that stimulated cells to proliferate for a short period of time. Then you performed your staining, so your S region would increase as much as G1 peak decrease.
In another situation, say a drug treatment that arrests cells in M phase (colcemid or mitomycin) if you wait a short time you would be in a similar scenario as the one descibed above. However if you wait enough, your G2M peak will increase until no G1 or S populations would be detactable.
Best,
Daniel
thanks Daniel, I perform flow after 72 h of treatment, where s increased, so maybe have to wait more for g2m increase to be visible?
Dear Muhammad Imran ,
This is a difficult question. To answer it, it would be good to know something about the type of cells, the treatment and the sampling for FCM, the protocol used to stain for nuclear DNA content. Assuming that you are staining cells in the context for their DNA content and that you measure intact cells for their DNA content, you will have the following options. If you just stimulate the prolifereation by increasing S-phase, this will temporarily increase G2/M phase, provided your sampling frequency is high enough. If you want to get an idea of the increase in DNA synthesising cells, you could add mitotic inhibitors such as colchemid, colchicine for animal cells and APM or oryzalin for plant cells at varing times after induction of the profileration. Since cells treated with these chemicals do not divide their chromosomes, they will end up as double DNA content nuclei in your FCM analysis. Experimental setup is very important in this case to have the proper control without treatment. If you are interested, I can provide you with a more detailed scheme how to tackle this problem. Kind regards, Harrie Verhoeven.
Some treatments may also just delay the time a cell needs to spend in S-phase without affecting the length (in time) of G2 or M. Then S will increase at the expense of both the G1 and the G2/M peak. Make sure you always gate out the doublets/cell aggregates when looking at cell cycle. PI-W (pulse width) against PI-A (pulse area) is the best, but at least use a scatter pulse geometry discrimination method if your instrument does not provide that option. G1 doublets create a false positive "G2/M cell" due to the double DNA content, so this is crucial. Best Regards, Idun Dale Rein
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