I am a PhD student working on glioma stem cells. I want to look at differences in proliferation rates between three glioma stem cell lines. I tried the CFSE assay where I incubated the cells with 10 uM dye for 15 minutes, changed the medium, incubated for an additional 30 minutes. At this time, I collected one sample of cells and fixed them for my t =0 control. The remaining three samples were incubated for 24, 48 and 72 hours, fixed and then analyzed by flow. I used U251 tumor cells which are known to be highly proliferative as a comparison.
I got nice sharp parent peaks in my t=0 control. So I think the staining works well. But when I used Modfit to analyze the curves it gave me proliferation indices for my different time points which do not seem to make sense. For U251 I got a proliferation . index of 1, 4, 10 for 0, 24 and 48 hours respectively and the population doubling time is known to be around 20 hours. But according to the proliferation index it shows that the cells have gone through four generations by 24 hours which is twice the reported doubling time in the literature.
Can somebody please help me make sense of the proliferation index. I used the t=0 as the control for ModFit. Do I need a different control?