Background: DNA analysis of endoreduplicating cells is difficult because of the overlap between stem-line G2 + M cells and 4C G1 cells. Simultaneous flow cytometry of DNA and cyclin B1 analytically separates these populations. The objective here was to develop simultaneous flow cytometry of DNA, cyclin B1, and p105 (highly expressed in mitosis) for improved, complete cell cycle phase fraction analysis of endoreduplicating cell populations.
Methods: Monoclonal antibody, GNS-1, reactive with human cyclin B1, was conjugated with fluorescein at three different fluorochrome-to-protein (F/P) ratios and tested for optimal sensitivity in a flow cytometric assay. A formaldehyde-methanol fixation procedure was optimized for retention of p105 within mitotic cells by analytic titration of formaldehyde. p105 was stained indirectly with Cy5-conjugated secondary antibody, followed by GNS-1, and DNA was stained with Hoechst 33342. The specificity of p105 in this assay was tested by comparison of manual and flow cytometric mitotic indices and by sorting and microscopic inspection.
Results: F/P 4.1 provided optimal fluorescein labeling of GNS-1. Formaldehyde (0.5%), followed by methanol permeabilization, fixed cells sufficiently to quantify stem-line and endoreduplicated G1, S, G2, and M phase fractions. Kinetic measurements of these fractions for both populations were demonstrated.
Conclusions: The fluorochrome-to-protein ratio is important and can be optimized objectively for these assays. A permeabilization-sensitive antigen (p105), previously requiring formaldehyde/detergent-fixed cell preparations, was shown to work equally well with formaldehyde/ methanol fixation. Three-laser, two-parameter intracellular antigen analysis can be successfully coupled with DNA content analysis. Cell cycle kinetic analysis of endoreduplicating populations should be improved.