QUANTITATIVE-ANALYSIS OF MITOTIC AND EARLY-G1 CELLS USING MONOCLONAL-ANTIBODIES AGAINST THE AF-2-PROTEIN

We have recently described a novel protein (AF-2), conserved between f ission yeast and man, and we have shown by flow cytometry (FCM) that A F-2 is highly accessible to specific monoclonal antibodies (MoAbs) in mitotic and postmitotic early-G1 phase cells. The aim of the present s tudy was to optimize the FCM methodology using MoAbs against AF-2 and to show that the evaluation of the mitotic cells, using different cell lines, was quantitative and reproducible. We found that a method base d on fixation with ethanol, instead of formalin, resulted in improved DNA histogram coefficients of variation and implemented separation of early-G1 cells from late-G1 cells. In addition, by eliminating several cell permeabilization and protein salt extraction steps, the method b ecame straightforward, conserved a clear-cut separation of the green f luorescence of M- with respect to G2-phase cells, and did not signific antly affect cellular integrity. The coefficient of correlation among the mitotic index values evaluated by this FCM method using MoAbs agai nst AF-2 and by microscopic visual counting was R = 0.94. When the FCM /AF-2 method was tested against an independent FCM method, which allow s clear separation of M- and G2-phase cells according to 90-degrees sc attering, we found R = 0.93. We conclude that MoAbs against the AF-2 p rotein may be used in FCM for quantitative analysis and for isolation of M-phase cells, providing as well, the identification of the early-G 1 cell subcompartment. The method may, in addition, be useful for the simultaneous detection of cytoplasmic cytokeratin and nuclear AF-2 ant igen.