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 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 degrees 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-G1 cell subcompartment. The method may, in addition, be useful for the simultaneous detection of cytoplasmic cytokeratin and nuclear AF-2 antigen.
Di Vinci
A
A
National Institute for Cancer Research (IST), Genoa, Italy.
Geido
E
E
Pfeffer
U
U
Vidali
G
G
Giaretti
W
W
D000911:Antibodies, Monoclonal / Q000276:immunology*; D015415:Biomarkers / Q000032:analysis; D002469:Cell Separation / Q000379:methods; D004247:DNA / Q000032:analysis; D000431:Ethanol / Q000494:pharmacology; D005404:Fixatives / Q000494:pharmacology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D007399:Interphase; D007633:Keratins / Q000032:analysis; D008214:Lymphocytes / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects; D008677:Metaphase; D008938:Mitosis; D008940:Mitotic Index; D009687:Nuclear Proteins / Q000032:analysis* / Q000276:immunology; D015203:Reproducibility of Results
D000911:Antibodies, Monoclonal; D015415:Biomarkers; D005404:Fixatives; D009687:Nuclear Proteins; D000431:Ethanol; D007633:Keratins; D004247:DNA