If you synchronize your cells, you will be able to monitor more than one cell cycle period

No need to worry about the dead cells. It can be effectively gate after you run the control cells.

In order tp exclude dead cells you can use a dye before fixing cells that will mark dead cells. After fixation the dye will be only in the fixed dead cells while the alive fixed cells will be unstained with this dye (see attached). You have to assure that for each condition you have approximately the same cell number for a good cell cycle analysis.

Don't need to worry about the maximum cell number limitation. You can always increase or decrease that arbitrary number when you acquire your cells in Canto II. Just keep consistent between the samples.

Hi!you should perform a time course with the same time points, more than two time points (12, 24, 48h, 72h etc) for both cell lines, keeping in mind their different doubling time. for each time point you should count death cells, and consider this data when performing FACS analysis. The forward scattering of the cells passed through the FACS instrument gives you an information about the death cells population, and you or some technician should be able to discard that population from your analysys.

You raise a number of important issues in your questions. First of all, if the net effect of adding growth factors is just to decrease the cell cycle time (hence faster growth rate), then you may find very little difference in the cell cycle distribution. Cell cycle distribution can only tell you if there is a significant difference in the fraction of cells in the different cell cycle phases, which may not change enough to be reliably detected. Fast-growing cell populations and slow-growing cell populations can look quite similar. Frederica Sardo is correct that you need to do different time points because the two cell lines have different cell cycle times and because the potential changes in cell cycle distribution may be (and probably are) transient.

The one million cell limit is not a hard limit but you can get dye-limited staining if you go much beyond that. You can simply add more stain, though, to get reliable results. One million cells is a good number to shoot for because it gives you plenty of cells to analyze. To get good cell cycle analysis, you need to run at least 50,000 cells and preferably about 100,000 cells, especially when you may be looking for small changes. That gives you much better statistics for the fit. And you have to be very careful about interpreting the fit using a good program like ModFit or MultiCycle. Perturbed cell cycles are always more difficult to reliably analzye. The other requirement is you need to have good CVs on the G1 peak to get good analysis. If you don't have that, your analysis will be meaningless. By good I mean ideally the CV would be less than 4%. If it is much greater than that you need to be sure an experienced person helps you. If it is more than 8%, forget it!

Regarding dead cells, you won't really be able to analyze them unless you dump off any floaters before you fix them. You have to fix the cells before staining them with PI (or use a detergent or hypotonic treatment) which means you can't distinguish between dead and live cells. Whether it matters depends on what information you need.

Finally, cell cycle analysis may not give you all of what you need. You certainly need to do it, but you should also do precise cell counts on timed samples so you get a good idea of the change in number of cells. You could also do a Tpot analysis but that more complicated and you need someone experienced with it to get good results.

I hope this helps.

I suggested that you can use the thymidine kinase 1 (TK; EC 2.7.2.21)- a cell cycle regulation of the enzyme, to check your results. The TK1 may provide an attractive system for your study because it is a kinase for which both positive and negative selections are available. We have previously published a general outline for counter flow centrifugal elutriation of cells (He Q., Wang N., Skog S., Ericsson S., Tribukait B. Characterization of a peptide antibody against a C-terminal part of human and mouse cytosolic thymidine kinase, which is a marker for cell proliferation. Europ. J. Cell Biol., 70: 117-124, 1996). Good luck.