Figure 1.
Plots indicating cell pathways through the cell cycle.
(a) A schematic indicating cell cycle routing of cells treated with ICRF-193 in fluorescence space (GFP-cyclin B1 signal as a function of DRAQ5 signal). In control conditions U-2 OS cells divide and remain in normal cycle (representing the proliferative fraction), with ICRF-193 treatment the U-2 OS cells continue to cycle however they bypass mitosis to enter a polyploid cycle (p) (representing the non-proliferative fraction). (b) Segmentation of experimental flow cytometry data representing molecular expression and DNA content used to initialise the CPM. Experimental data at texpt = 0: experiment non-gated data (green and red markers), experimental gated data/initial virtual population (red markers) and the dashed black line refer to the contour defining the gated data. The solid black line indicates the best representation of the fitted population (see text).
Figure 2.
Flowchart indicating the main steps of the cell population model (CPM).
Figure 3.
Comparison of traditional analyses of static flow cytometric data and to that used to develop dynamic data analysis.
(a) Displays the fluorescent frequency histogram of the DRAQ5 intensity, plots (b) and (c) graphically illustrate how the median DRAQ5 and GFP cyclin-B1 fluorescence signals vary as a function of a normalised intermitotic time (light grey and blue lines respectively. Insert: plot (b) - magnification of the polynomial fit to the frequency histogram of the DRAQ5 intensity.
Figure 4.
Model simulation to track a virtual sub-population (red markers) of vcells through the cell cycle at (a) 6 hours (b) 12 hours (c) 18 hours and (d) 24 hours.
Note: the overall vcell population has a mean intermitotic time of 22 hours and an associated standard deviation of 6 hours.
Figure 5.
Endocycle routing with ICRF-193 (a), (b) and (c) indicate experimental data (green markers), gated sub-set (red markers) and a contour of the gated data (dashed black line) at 0, 24 and 42 hours respectively.
Figure 6.
Simulated vpopulation at (a) 24 and (b) 42 hours respectively.
The red shaded areas are virtual cells that lie within the respective experimental gated contours shown in Figures 6(b) and (c), the green shaded points are simulated data lying outside of these contours. Also indicated is the gated, time zero experimental data set contour (black dashed line).
Table 1.
Optimised parameter values of the vcell population.
Figure 7.
The normalised virtual cell count throughout the time-course of the experiment.
The blue, green, red and yellow curves indicate the fraction of virtual cells in the 2n, 4n, 4np and 8np phases of the cell cycle respectively. In addition, the fraction of virtual cells drug blocked throughout the simulated experiment is indicated by the black curve.