Production of ROS in the presence of anti-D–opsonized RBCs. Bar chart shows the production of ROS and how they vary with increasing concentrations of anti-D–sensitized RBCs in monocytes (□) and granulocytes (▪). The inset represents ROS production in control tubes containing the particulate stimulus E coli, the low stimulus fMLP (N-formyl Met-Leu-Phe), and the high stimulus phorbol 12-myristate 13-acetate (PMA). ROS were determined by co-incubating different ratios of WBCs with anti-D–coated RBCs for 10 minutes and measuring the oxidation of the fluorophore dihydrorhodamine (DHR) 123 by flow cytometry. The data are expressed as fold change in ROS production and calculated by the formula: percentage of fluorescence (FL1)–stimulated cells/percentage of fluorescence (FL1)–nonstimulated cells. The results are shown as the means ± SDs of 5 independent experiments. Significance values are indicated (*P < .005; **P < .0001).