NOX2 is important for neutrophil-platelet aggregation under stirring conditions and for hepatic ischemia/reperfusion injury in vivo. (A-B) In vitro neutrophil-platelet aggregation assay was performed as described in the “Methods” section. Neutrophils and platelets isolated from WT and NOX2 KO mice were labeled with Alexa Fluor 647-conjugated anti-Gr-1 and Dylight 488-conjugated anti-CD42c antibodies, respectively. Thrombin-activated platelets were mixed with TNF-α-treated neutrophils under stirring conditions (1000 rpm). Cells were analyzed by flow cytometry. R1 designates leukocyte-platelet aggregates and R2 designates total population of neutrophils. Neutrophil-platelet aggregation was measured by the fluorescence intensity of anti-CD42c antibodies (B) in the R1 gate. Data represent the mean ± SD (n = 3). SS, side scatter; FS, forward scatter. (C-F) Hepatic ischemia/reperfusion injury was induced as described in the “Methods” section. (C) Representative pictures of liver sections stained with triphenyltetrazolium chloride. (D) Infarct sizes (white areas in C) were measured by Image J. (E) Serum levels of aspartate aminotransferase (AST). (F) Neutrophils (arrow heads) and platelets (arrows) in liver sections were stained with an esterase kit (pink) and anti-CD41 antibodies (brown), respectively. Nucleated cells were stained with hematoxylin (purple). Bar = 20 μm. (G) The number of neutrophils was counted inside (gray) and outside (white) the hepatic vessels. (H) The number of platelets that interact with adherent neutrophils to the vessels was counted. Data represent the mean ± SD (n = 5 mice and 15 sections in 5 mice for G-H). *P < .05, **P < .01, or ***P < .001 vs WT control or sham after ANOVA and Dunnett’s test. In (G), **P < .01 was obtained after comparison of the total number of neutrophils. #P < .05, ##P < .01, and ###P < .001 between 2 groups after Student t test.