"Cell cycle flow cytometry" question (again):
Since results are discriminated in diploids and tetra or aneuploids, how should I interpret these values? I explain: most of the cells are determined to be diploid and these are divided in G1, S or G2/M phase. And, approximately, 1/10 of total events is referred to as tetraploid or aneuploid. How should one interpret this minority category of cells?
First, you should make sure that these cells are actually tetraploid and not doublets. You can do that with the doublet discrimination module (DDM) in CellQuest, after passing your cells through a 70µm mesh.
If they are still there, you can now try to find out what they are. Chromosome spreads can help you, and also depending on what you did to these cells (and what cell type) you can anticipate what happened: re-replication, endoreplication, endomitosis, cell fusion...
First, you should make sure that these cells are actually tetraploid and not doublets. You can do that with the doublet discrimination module (DDM) in CellQuest, after passing your cells through a 70µm mesh.
If they are still there, you can now try to find out what they are. Chromosome spreads can help you, and also depending on what you did to these cells (and what cell type) you can anticipate what happened: re-replication, endoreplication, endomitosis, cell fusion...
To use one of software programs to evaluate such as WinCycle or ModFit will be helpful for interpretation. I hope you have used a negative and a positive control so that you can be sure that your sample is in good condition and tetraploid population is a real one. You can check with V. Coulon's suggestion or have a positive cell line that has a tetraploid population.
If you are sure that your results are not an artifact and you really have tetraploid or/and aneuploid cells but not doublets, triplets and so on (and you are not able or do not want to use different cell line) you may perform a little bit more complicated double parameter cell cycle analysis using as a second parameter one of proliferation-associated proteins. The most widely used markers are the proliferating cell nuclear antigen (PCNA), the antigen detected by Ki-67 antibody, cyclins A, B, E, D and so on. You can easy find a lot of articles with descriptions of the methods and many markers in sale. I do not know exactly your tasks, but it is possible that you will be able to register the same of even different response of the diploid and tetraploid cells to the respective treatment (some cell lines after specific treatment may change ploidy, so you may get as a result only diploid or only teraploid populations). In both cases it will be more interesting than just to discriminate such cells and forget about them. But, first you need to be sure, that you are dealing with the suspension of single cells.
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