Jill Martin wrote: > . . . I have an investigator who is looking at proliferation and would like > to see the uptake of Brdu in S-phase. We would like to do an accurate cell > cycle analysis which will give us not only the percentage of each phase but > the exact boundaries so that we can gate on them. Cell cycle fitting programs, like ModFit (verity) or MultiCycle (Phoenix) will fit your DNA histogram data and effectively define limits of your cycle compartments. These limits can then be transferred as gating criteria. > It would be nice to be able to plot this histogram with a two dimensional > contour plot. Most of the plots in the literature show a nice horseshoe > shaped curve but there is no indication exactly where one phase ends and > another starts. The incorporation of BrdU by definition reveals S-phase cells, so setting limits in a 2-D plot of PI vs BrdU such that the negative control (no BrdU added) shows no/low events above a specified limit should allow you to specifically detect S-phase (BrdU incorporating) cells as those attaining and surpassing this limit. The observation that some of the BrdU positive cells are in line with G1 or Mitotic DNA level cells is probably more an artifact of the PI staining technique (its inability to efficiently resolve these compartments from S-phase -- which can be improved as you improve the % c.v. of your peaks). If you log amplify your BrdU signal, this becomes more evident since low signal cells (low or non-specific incorporation) are better resolved. > Ideally we would like the software for Mac, but we could manage to use a > P.C. if necassary. In a perfect world, any software package you use should have the ability to correlate DNA histogram fitting with bivariate analysis. This unfortunately (as far as I know, anyway) is not generally the case.I think Verity's WinList combines these capabilities . . . most others have different companion programs to do these analyses in parallel. WinList is now available for both MAC and PC. Phoenix Flow Systems sells several programs, and it looks like they provide integrated cell cycle analysis as well. Their programs operate in a DOS or Windows environment. FLOWJO also provide this combined analysis capability. on the MAC only. All of these programs cost (some bigger than others, but mostly big money). Some programs available as shareware (WinMDI as example) will do the necessary bivariate analysis necessary for enumerating BrdU incorporating cells. Perhaps there are others that can be discussed . . . MAK. -- Mark A. KuKuruga, Managing Director University of Michigan Core Flow Cytometry kukuruga@medmail.med.umich.edu
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