EFFECTS OF FIXATION METHOD ON IMAGE CYTOMETRIC MEASUREMENT OF DNA CONTENT AND DISTRIBUTION IN CELLS STAINED FOR FLUORESCENCE WITH PROPIDIUMIODIDE

We performed image cytometric measurements of DNA content and distribu tion on cycling human HCT-8 cells stained for fluorescence with propid ium iodide (PI). Seven different fixation protocols were evaluated for stoichiometry of PI staining and for their ability to preserve in viv o chromatin structure. Bimodal integrated optical intensity (IOI) hist ograms were obtained with all fixation protocols. Increased accessibil ity of DNA to the dye was evident in increased values of the IOI at th e GI peak. The fixatives studied, in order of increasing accessibility to DNA, were Regaud's, Boehm-Sprenger, Cornoy's, air-drying, methanol , ethanol, and acetone/methanol. In general, the coefficient of variat ion of the IOI within the G1 peak was higher for fixatives where DNA i s less accessible. Features describing the spatial distribution of sta in exhibited dramatic changes for Boehm-Sprenger fixation, which were consistent with the observation that in vivo conformation of chromatin is best preserved with this method.