ROS is required for HIT IgG-induced NETosis. (A) Representative histograms of DiOC₆ fluorescence in the neutrophil (CD15⁺) population when stimulated with normal IgG (orange), KKO (left), or HIT IgG (right) plus heparin (0.5 U/mL) in the absence (red) or presence (blue) of DPI, using flow cytometry. (B) Quantification of mitochondrial membrane potential in control or HIT immune complex–activated neutrophils from healthy individuals, with and without NOX inhibitor DPI (n = 4). (C) Representative histograms of DIH123 staining in the neutrophil (CD15⁺) population when stimulated with normal IgG (orange), KKO (left), or HIT IgG (right) in the absence (red) or presence (blue) of DPI. (D) Quantification of ROS production in control or HIT immune complex–activated neutrophils from healthy individuals, with and without NOX inhibitor DPI (n = 4 except HIT3, n = 3). Representative dot plots of (E) NETosis (MPO⁺Cit H3⁺) and (F) the LDG population (CD15⁺ events, white dots). Quantification of (G) percentage of LDG (n = 3) and (H) NETosis in blood treated with control or HIT IgG complexes, in the absence and presence of DPI (n = 4). Data expressed as mean plus or minus SEM. *P < .05, **P < .01, ***P < .001, and **** P < .0001 by Student t test . FSC, forward scatter; MPO-PE, myeloperoxidase-phycoerythrin; Ms IgG, mouse IgG (used as control for KKO); Norm, normal; SSC, side scatter.