Fig. 2. Multi-parameter flow cytometry analysis of MT content of G2- and M-phase subpopulations. K562 cell lines harboring pMEP-vector (Control) or pMEP-MAP4 (MAP4) were analyzed after 20 hours of Cd²⁺-induced expression. One culture of control transfected cells was treated with Paclitaxel (50 ng/ml for 20 hours). Cells were extracted with a MT-stabilizing buffer and fixed according to a protocol designed for determination of MT-specific fluorescence (see Materials and Methods). (A) Cells stained with a histone H3 phosphoepitope antibody were analyzed with respect to propidium iodide-stained DNA and 90° side-scattering properties by dual parameter flow cytometric analysis. This analysis allowed definition of the G1-, G2- and M-phase gates indicated in the dot-plots. (B) Determination of the fractions of histone H3 phosphoepitope-positive cells within the G2- and M-phase gates defined in panel A. (C) Distribution of MT-specific fluorescence within the G2-populations and M-populations (dotted line). Staining of cells with fluorescein-conjugated rabbit anti-mouse immunoglobulin alone gave <1% nonspecific staining (not shown). (D) Mean MT-specific fluorescence intensities of G1-, G2- and M-phase populations derived by gating of cells as depicted in panel A. More than 95% of all cells were included in the acquisition gate and the data are representative of three independent transfection experiments.