Quantitative analysis of mitotic and early-G1 cells using monoclonal antibodies against the AF-2 protein

A. Di Vinci, E. Geido, U. Pfeffer, G. Vidali, W. Giaretti

Research output: Contribution to journalArticle


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 MoASbs 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 permeabilizationa 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 evaluted by ths 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° scatering, 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-G1 cell subcompartment. The method may, in addition, be useful for the simultaneous detection of cytoplasmic cytokeratin and nuclear AF-2 antigen.

Original languageEnglish
Pages (from-to)421-427
Number of pages7
Issue number4
Publication statusPublished - 1993


  • Flow cytometry
  • Mitosis
  • Proliferation associated antigen

ASJC Scopus subject areas

  • Biophysics
  • Cell Biology
  • Endocrinology
  • Hematology
  • Pathology and Forensic Medicine

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