I have measured cell cycle progression of my cells by staining my cells with propidium iodine (PI). I obtained the histogram unlike what is expected with a high abundance of S phase in control cells and more cells in G2/M phase that expected.
It is the first flow cytometry experiment I did so I am wondering if I did something wrong to receive these data (protocol attached). Do you think it might have something to do with the cells handling?
I am using HEK293 cells but our Flow Cytometer (FC500 from Beckman Coulter) is mostly set up for the yeasts. We do not have any program set for the human cells and only one colleague of mine adjusted the program for HeLa cells.
I would really appreciate your feedback. Thank you in advance.
You may check your first two gates that you applied regarding selection of cells based on size and scatter (FS vs SS) and singlets (FS Area vs FS Width).
You can also apply singlet gates on your PI channel that is FL4.
If you are leaving out some relevant cell population, that might explain the observed data.
Best
S
Thank you for your answer but I am afraid that does not account for my situation. Ungated data looks almost identical. With my gating I only removed dublets.
Ohh OK.
In that case you would have to accept the data.
It may be the case that at the time you harvested the cells they were actively growing, hence more population in S and G2M compared to G0/G1.
You may try harvesting cells at different intervals post passaging and see if the data changes.
Cell cycle labelling should work with PI as long as the membranes are appropriately permeabilised. Your cell cycle profiles suggest that you have a good fixation/staining procedure. I can't see how many cells you have recorded but the profiles suggest several thousand so they are smooth and will have high statistical strength. It appears that you have cells in to phases - your results active proliferation of cells in log growth(like a tumour line) while the Expected profile looks like naive PBMNC were the majority of cells are in G0/1(Quiescent state). Both look like normal profiles. Try your procedure on some fresh PBMC,
or see what happens to the cell cycle in HEK that are overgrown , were I would expect cell:cell inhibition to reduce the S and G2/M contribution, as cells drop out of active cell progression. Or try a metabolic inhibitor eg cytochalasin D, Indisulam, ... to confirm your labelling is correct for actively proliferating cells
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