Flowers, ModFit software from Verity has a "Proliferation Wizard" that does all the proliferation calculations for you from cells stained with CFSE or a PKH dye (for example, it gives you the precursor frequencies of the proliferating cells and it also gives you a proliferation index that tells you how many more cells you have now than when you started the culture). What ModFit does is model the intensity histogram of the cells --- so it can use the separate or quasi-separate peaks found in some cases with CFSE-stained cells. Or it models the theoretical positions of the peaks for dividing cells if you are staining with the PKH dyes (that don't give such good intensity separation) or if your CFSE staining hasn't given separate peaks. Main problem with ModFit software is that it is using the theoretical position for the intensity peaks of the proliferating cells -- it won't be as accurate if cells are dividing asymmetrically and the proliferation peaks don't match the theoretical positions expected for halving of intensity with each division. We have found that it matches the peaks quite well with CFSE -- as long as you use a correction for the particular amplifcation factor for your log amplifier (the log decades full scale are hardly ever exactly 4). And we have also found that the ModFit software gave us values that correlated well with the known values of a model system. Conflict of interest: After writing our paper on using PKH and flow to quantitate precursor frequencies of antigen-specific T cells (J. Immunol. Methods 230: 99, 1999), I worked with Verity to get them to include the precursor frequency calculation in their ModFit software. I have no financial interest at all in this --- but am somewhat devoted to the concept of using CFSE or PKH to do precursor frequency calculations as I feel this method provides much more complete analysis of proliferative responses than does tritiated thymidine (which is only a bulk assay related to the total number of proliferating cells at the time of the assay). Others who, before us, have used CFSE and flow to quantitate precursor frequencies are Wells et al (J. Clin. Invest. 100: 3173, 1997) and Song et al (J. Immunol. 162: 2467, 1999). If I am missing any references on this, please let me know. Alice Alice L. Givan Englert Cell Analysis Laboratory of the Norris Cotton Cancer Center Dartmouth Medical School Lebanon, New Hampshire NH 03756 tel 603-650-7661 fax 603-650-6130 givan@dartmouth.edu
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