Flow cytometric analysis of ZAP-70 expression and flow cytometric profiles of ZAP-70 protein on 4 B-CLL cases. (A) The method used to quantify the expression of ZAP-70 by B-CLL cells is shown. In plot 1, forward and side scatter of mononuclear cells is represented with a region (R1) drawn around the lymphocytes. Lymphocytes were R1 gated, and then T cells and natural killer (CD3⁺CD56⁺) cells (the R2 region in plot 2) and CLL cells (CD5⁺CD3^–CD56^–) cells (the R3 region in plot 3) were selected according to their phenotype. The plot 4 shows the expression of mouse IgG1 isotypic controls after lymphocyte (R1) gating. The plot 5 shows the expression of ZAP-70 after lymphocyte (R1) gating. For the purpose of quantification, markers were placed so that the T cells and natural killer cells (R1 and R2 gated) with a high level of expression of ZAP-70 would appear in the upper-right quadrant (plot 6). Mouse IgG1 isotypic antibody conjugated with Alexa Fluor was used as control marker for ZAP-70 positivity. Then, B-CLL cells were plotted, and the same marker that included T cells and natural killer cells in the upper-right quadrant was used to calculate the percentage of CLL cells that were positive for ZAP-70, as shown in the plot 7. Panel B shows the level of ZAP-70 expression by lymphocytes from 4 representative patients with CLL according to the mutational status of IgV_H status. In each case, mouse IgG1 isotypic antibody is used as control for ZAP-70 positivity. The percentage of CLL cells with a high level of ZAP-70 expression is shown in the lower-right quadrant of each plot, after the exclusion of T and natural killer cells. FITC denotes Alexa Fluor 488.