Figure 2

The expression of c-mpl is significantly higher in stage I CD34⁺ HSC/PC and decreases with advancing differentiation stage. A&B. For each specimen from every tissue source, CD34⁺ cells were plotted with FlowJo^® software on a CD34-FITC vs. CD38-PE contour plot, and gates demarcating HSC/PC differentiation stages I through IV [6] were applied (as depicted in Fig. 1A). Cells within each stage were stained with an IgG1 isotype control-APC antibody equivalent in concentration to the c-mpl-APC antibody used. On a log histogram plot of IgG1-APC fluorescence, a c-mpl^-- gate was manually drawn to include the left-most 99.5% (± 0.2%) of isotype control-stained cells. A c-mpl⁺ gate was manually defined to extend from the end of the c-mpl^-- gate to the far right of the histogram plot. These gates were defined separately for each HSC/PC differentiation stage for each tissue source, and then applied without alteration to their corresponding c-mpl-APC stained cells within each stage from each tissue. The percentage of cells falling within the c-mpl⁺ gate was then calculated by the FlowJo^® software. Error bars = ± standard error of mean (SEM). C&D. For every specimen, the median fluorescent intensity (MFI) of viable CD34⁺ cells from each differentiation stage was calculated by FlowJo^® software from the log histogram plot of the c-mpl-APC fluorescence parameter. These MFI values for each stage of each tissue specimen were then normalized individually with the appropriate MFI value of the corresponding differentiation stage HSC/PCs stained with the IgG1 isotype control, yielding the Relative MFI (RMFI). Error bars = ± SEM.