dear group, I have a user who is trying to synchronize different cell lines in G1 to perform electrophoretic mobility shift assays on those cells. most of the lines can be synchronized to 90-95% G1 (by ModFit LT) by serum starvation. one is being recalcitrant with about 85% in G1 and 15% in G2/M, although earlier it was behaving as well. she feels she must have >90% in G1 for her assays. in an effort to improve the situation, she ordered a new stock of the cell line (MFC-10) and started over. they didn't work well either. but they are also doing something that doesn't make a lot of sense to me. since we run some, but not a whole lot of PI-stained samples, i am stumped. in a plot of PI-width vs. PI-area, the cells are now spreading out horizontally, showing a great variation in PI-width. microscopically, they are very heterogeneous in size. but on the width vs. area plot, there is also a slope to the line of cells in G1 and those in G2/M; there is a slight increase in PI-area associated with the increase in PI-width. My questions are: 1. could differences in chromatin condensation be responsible for this correlation? if not, what else? is there any way to fix this with her cells? 2. how do i model this situation? the effect is to increase the CV of the G1 peak on the PI-area histogram to about 5-6%. visually, the width vs. area dot plot is very tight. it seems that i could use WinList to do software "compensation," but somehow that feels wrong in this situation. and i am not sure that WinList's compensation algorithm compensation will model this type of correlation. still, it is a mathematical remedy for a biological predicament, and that makes me uncomfortable. oh, and she is saturating the DNA with PI, because she has even tried centrifuging the cells, resuspending in the PI stain and re-incubating overnight. the plots are the same thanks in advance, Roger Smith Dept. Veterinary Pathobiology Texas A&M University Phone: (409) 845-5167 Fax: (409) 862-1088 e-mail: rosmith@cvm.tamu.edu
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