Open AccessQuantitative analysis of mitotic and early‐G1 cells using monoclonal antibodies against the AF‐2 protein
Author(s) -
Di Vinci Angela,
Geido Elio,
Pfeffer Ulrich,
Vidali Giorgio,
Giaretti Walter
Publication year - 1993
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/cyto.990140411
Subject(s) - monoclonal antibody , flow cytometry , mitosis , biology , microbiology and biotechnology , cell cycle , cell culture , cell , antibody , biochemistry , immunology , genetics
We have recently described a novel protein (AF‐2), conserved between fission yeast and man, and we have shown by flow cytometry (FCM) that AF‐2 is highly accessible to specific monoclonal antibodies (MoAbs) in mitotic and postmitotic early‐G1 phase cells. The aim of the present study 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 based 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 became straightforward, conserved a clear‐cut separation of the green fluorescence of M‐ with respect to G2‐phase cells, and did not significantly affect cellular integrity. The coefficient of correlation among the mitotic index values evaluated by this FCM method using MoAbs against 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 allows clear separation of M‐ and G2‐phase cells according to 90° scattering, we found R=0.93. We conclude that MoAbs against the AF‐2 protein may be used in FCM for quantitative analysis and for isolation of M‐phase cells, providing as well, the identification of the early‐Gl cell subcompartment. The method may, in addition, be useful for the simultaneous detection of cytoplasmic cytokeratin and nuclear AF‐2 antigen. © 1993 Wiley‐Liss, Inc.