Alice is correct that the ModFit LT Proliferation Wizard is designed to work with indistinct populations frequently seen when doing PKH or CFSE analysis. The theoretical positions she describes are the calculated mean position of the daughter populations. These positions are calculated using information about the log decades in your system. By default this calculation is based on 4-log decades which gives a theoretical position shift of -19.19 channels for each daughter population. Changing the default setting is as simple as entering the actual log decades of your instrument. The software then adjusts all the calculations for the daughter populations. ModFit LT also has the capability of automatically calculating asymmetric cytokinesis. This function is accessed through the Proliferation Wizard. In some cases histograms clearly show an asymmetrical pattern with respect to the shift of the daughter populations. We know that the CFSE samples can be affected by auto-fluorescence which shifts weakly fluorescing daughter populations upscale. We also know the analysis can be affected by the characteristics of the log amplifier. If the log amplifier has irregularities in its signal response it could result in irregular shifts in daughter populations. The only way to truly know the cause of these shifts is to perform a careful analysis of the log amplifier's characteristics. Once these characteristics are known the standard proliferation model can be adjusted. There have also been several occasions where customers have supplied data showing distinct PKH and CFSE populations. Using the flexibility of ModFit LT's model editor we were able to provide these customers with models that automatically adjusted for the asymmetrical nature of these histograms. As many of our customers will attest, we are glad to assist any ModFit LT user with these special needs. Best Regards Don Donald J. Herbert Technical Support Manager Verity Software House, Inc. PO Box 247 45A Augusta Road Topsham, ME, USA 04086 Phone: (207) 729-6767 ext.190 Fax: (207) 729-5443 email: tech@vsh.com web: www.vsh.com -----Original Message----- From: Alice L. Givan [mailto:Alice.L.Givan@dartmouth.edu] Sent: Friday, August 09, 2002 9:36 AM To: cyto-inbox Subject: Re: query on LPAs: replacing tritiated thymidine with flow 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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