We detect the cell apoptosis and cycle of A549 affected by chemical compound through flow cytometry (C6, BD inc.). The results are shown in the attachment, our questions are as following:
1. What's the upper left (UL) quadrant represent in the apopsis result ?
2. In the apopsis result，why cell content in UL increased from A to 2A but decreased in 5A ?
3. In the cycle result，Why the peak of cell move to the left from A to 2A ?
Hello Zhe,
To 1.: This is not the usual way to label you axes. Ususally you have Annexin-V on the x-axis and PI on the y. In the plot you show above UL are Annexin-V positive, that is, "early apoptotic" cells. Flip you axes and you'll be able to compare your results to the literature.
To 2.: First see above. It may depend on the time point. For sure it seems like 5A gets you a much stronger kill, with fragmentation of the nuclear membrane so PI can enter to stain the DNA. Not too unususal I'd say. Try the same experiment with a shorter incubation.
To 3.: Your axes are labelled incorrectly! x should be PI and y should be cell number. Your drug my very well be influencing PI fluorescence. One way yould be to adjust voltage to keep you S1 peak in position. Definitely run some control experiments checking for this effect of your drug. E.g. Short term treatment that can not possibly have an effect on the cell cycle and then measure.
Hope that helps.
3:
From my experience, I would say the result in 2A should be normal while the others are problematic (if you didn't change the voltage setting between different samples). If you look more carefully, the peaks in the histograms of the control, A and 1.5A are broader than that in 2A, together with a shift to the right-hand-side for all of the events. Moreover, the severity of the problem decrease with increasing drug concentration. I would say this is related to the incomplete digestion of RNA in your samples. The more drug you add, the fewer cells you have. When you have more cells in the control and also lower drug concentrations, there are more RNA which is not completely digested and being stained by propidium iodide, leading to a rightward shift and also the broadening of the peaks. Try adding a bit more RNase to your samples and allow a longer digestion.
Moreover, if the drug causes apoptosis, you can also try lowering the threshold value so that you can see also the sub-G1 peak.
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