Questions about the Sub G1 peak in the cell cycle distribution analysis.
Significant late apoptotic positive results were obtained when the cells were assayed for apoptosis using Annexin V / PI double staining (BD Pharmingen, Oakville, ON, Canada) by using Accuri C6 flow cytometer, but the sub G1 peak was not observed at the cell cycle test by PI staining, The inconsistencies in the results of these two experiments make it difficult to explain.
My main concern is if the methods we used to test the sub G1 peak is inappropriate. Alternatively, we assume that the results is reliable, and an explain is needed.
fig 1. is about how I analyzed the data included the way I gated(I did not select cell debris because I do not think debris can be seen as "cell") I wonder whether I have make the correct choice.
fig 2. is the result of the Annexin V / PI double staining, Annexin V+PI+ is the late apoptosis stage. the result is obviously positive. So I wonder why the sub G1 peak did not appear.
And I I don't know if the PI staining buffer I used is correct(did I missing some components?):
pbs:1.8ml
RNase:40μl
Triton-100:10μl
PI(10mg/ml):150μl
critric acid monohydrate:2mg
fig 3 is control(compared with treated cell(fig 1))
about fig1 vs fig3, what I wonder is that should I select cell debris in the gate in the FSC/SSC?
Hi,
If you share your protocols and software adjustments for both assays with results, one may help to explain.
Hi Zhe, according to your data, there are probably several explanations:
1. How did you define the "late apoptosis stage"? Annexin V+PI+ or only PI+? The thing is this: during apoptosis, the cells become reactive with Annexin V after the onset of chromatin condensation but prior to the loss of the plasma membrane's ability to exclude PI; however sub-G1 peak represents a factional DNA content, which means the cellular DNA has been degraded within apoptotic cells in a very late apoptotic stage, and the fraction of DNA leaks out during cell staining. One possible reason is that there might be a gap between you-defined late apoptosis stage and the final DNA degradation, which leads to a positive result from Annexin V/PI staining but a negative result of sub-G1 peak.
2. Another possible reason that can be overlooked is the gating that used in your FACS analysis. If you want to analyze sub-G1 peak, you should gate all the cells including those debris-like cells which are usually strictly gated out during analysis.
3. Moreover, I don't think sub-G1 peak is a good indicator for cell apoptosis, since any fractional DNA content, not only caused by apoptosis but also by other reasons, can result in multiple nuclear fragments. For example, mechanically damaged cells, cells with different chromatin structure, even unfixed cells which are lysed in hypotonic solution. Thus, sub-G1 peak cannot fully represent the number of apoptotic cells. Why don't you try other methods to double check your results, such as Caspase 3/7 assays, TUNEL, etc.
Hope this information are helpful for you!
I would recommend added data obtained to make it easier for us to help you interpret your results. Han gives some excellent points, but I don't think you have provided enough information for us to make appropriate suggestions.
fig 1. is about how I analyzed the data included the way I gated(I did not select cell debris because I do not think debris can be seen as "cell") I wonder whether I have make the correct choice.
fig 2. is the result of the Annexin V / PI double staining, Annexin V+PI+ is the late apoptosis stage. the result is obviously positive. So I wonder why the sub G1 peak did not appear.
Hi Zhe Zhang,
I agree with Anne, because we have to see your control vs treated samples to evaluate the data.
By the way, I totally agree with Han. The gating range must be increased.
The collection of the cells is also important. If you discard the culture medium which possible have late apoptotic cells or DNA debris (SubG1 components), you can never see the SubG1.
When I look at the first figure, your gating looks correct for the first two dot plots. Your histogram shows the smallest peak in the sub G1 but it is rather minimal at best. (You can even see the three separate populations in the second dot plot that are depicted in the third histogram.)
Cigdem makes a great point: how you collect the cells is very important. If you are trying to look at both living and dead cells, make sure you aren't discarding that dead population. Dying or dead cells are buoyant in nature and take a little extra time to pellet down during harvesting.
The subG1 peak represents pieces of cells. In fact, each cell can result in the formation of several fragments. Simply cleaving DNA during apoptosis to make the histone ladders doesn't actually deplete the DNA - you don't actually have to lose that much DNA binding capacity. The degradation of the cells may involve longer kinetics, if you really want to see those small pieces - but it looks like you have it optimized to detect apoptosis. Also, don't forget that there are different ways of inducing apoptosis, and certain inhibitors might actually cause apoptosis but inhibit the subsequent blebbing and disassembly of the cell. As mentioned above, we don't know the cells or the treatment, so we can only talk in general terms. But I would not necessarily have significant concerns if that data were generated in my lab - it all depends on the conditions and the timing. An easy way to solve it is to simply confirm via other ways - and this is always a good idea. PARP cleavage via immunoblot, histone ladder gels, comet assays could all help to make your point.
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