I am working on primary mice sertoli cells. I supplemented sertoli cells with different concentraions of a hormone for 48hrs. That drug cause apoptosis in conc. dependent manner check by both protein and mRNa level, but there is no siginfiant difference in the cell cycle assay. So my question is; is it possible that there is no significant change inthe cell cycle phases? or some thing wrong in my experiment etcn., I need guidance please, Thank you.
You do not provide specifics of the assays that you had performed, so I just assume that you analyzed cell cycle distribution using intercalating dye (PI) to stain permeabilized cells and flow cytometry analysis. I also assume that you included to analysis (gated in) all relevant cells except for cell debris and cell aggregates. In this case, DNA fragmentation in apoptotic cells should result in detectable sub-G1 cell population, so the distribution of cells in cell cycle phases should change.
If your setting of PMT prevented you to acquire signals of sub-G1 cells (or if you gated them out), you may have seen no change in cell cycle distribution even if your treatment induced apoptosis, provided that the apoptosis was induced equally in all cell sub-populations (G0/G1, S, G2/M).
Likewise, the sub-G1 peak corresponding to apoptotic cells may not appear, if cells enter apoptosis from S or G2/M phase of the cell cycle. However, in this case, the proportions of cell cycle phases would still be different between treated and control cells even without sub-G1 area appearing in the histogram.
In conclusion, it is possible to induce apoptosis and observe no change in proportions of cell cycle phases, if one misses the sub-G1 area and the same proportion of apoptosis was induced in cells from different cell cycle phases.
@Roman Mezencev: Thank you you very much for detailed answer, really it can slove my confusion.
Except for sub-G1 issue, single PI staining for cell cycle analysis is not very reliable. Firstly, it is very difficult to gate in slow proliferating cells which show small S and G2/M peak; and many drug treatments will change DNA intercalating capacity, so will shift all the peaks. Meanwhile, there is no chance to discern change in G2/M transition by PI staining.
To avoid this problems, Edu+PI+p-Histone H3 staining can detect all 4 cell cycle phases without any ambiguity.
@Xiong, Thank you vey much for the informations. Actually I am working on sertoli cell and they are fast growing cells in vitro culture. so think PI stainig is suitable to use in FAC. Still your suggestion is helpful for my research. thanks
@Patrick Aouad. Yeah, Firstly I checked apoptosis by flowcytomtery confirmed by Western blot and then qPCR. Obviously I also checked proliferation assay by kit and followed by counting method supported by proliferation marker through qPCR. These results show that the drug causes apoptosis in dose dependent manner in sertoli cells.
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