Production of ROS in SS RBC is mediated in part by NADPH oxidase activity. (A) A schematic depiction of ROS metabolism along with inhibitors of enzymatic sources of ROS. (B) The ROS signal in SS RBC is reduced by preincubation with the NADPH oxidase inhibitor DPI (10 μM) prior to staining with CM-H2-DCFDA, but not by the xanthine oxidase inhibitor oxypurinol (500 µM) or the mitochondrial electron transport inhibitor rotenone (50 µM) (n = 6). (C) The ROS signal in SS RBC is reduced by preincubation with any of the known inhibitors of NADPH oxidase (DPI at 50 µM, gp91-dsTat at 50 µM, and apocynin at 100 µM), supporting the involvement of NADPH oxidase in ROS generation in these cells (n = 3 and P < .05 where designated by asterisks). (D) Whole-blood samples from AA and SS subjects were colabeled with PE-Cy7 anti-CD45, PE–anti-GPA, and CM-H2-DCFDA to permit the identification of WBC (CD45⁺;GPA⁻) and RBC (CD45⁻;GPA⁺) populations and the quantitation of the total ROS signal from each population (shown in the histograms as red for the RBC population, blue [thin line along the x-axis] for WBC and purple for the total RBC + WBC populations, in representative samples). (E) RBC ROS production, identified by gating out the CD45⁺ WBC population, constituted almost 80% of the combined RBC and WBC ROS signal. In AA samples (n = 10), the signal from RBC alone was 79% ± 1.5% of the total signal, whereas in SS samples (n = 7) this proportion was 77% ± 4.7%. FSC, forward light scatter; SSC, side light scatter.