ROS generated from neutrophil NOX2 are crucial for the activation and ligand-binding activity of αMβ2 integrin. (A-B) Mouse neutrophils were treated with or without fMLF, and the surface expression of PSGL-1 and αMβ2 integrin was measured by flow cytometry. (C) WT and NOX2 KO neutrophils were pretreated with or without 1 μM H₂O₂ and stimulated with fMLF. Lysates were immunoprecipitated (IP) with control IgG or anti-β2 antibodies, followed by immunoblotting and densitometry. (D) Human neutrophils pretreated with 1000 U/mL catalase were stimulated with fMLF, followed by flow cytometry using antibodies against total (ICRF44) and activated αMβ2 (CBRM1/5). Data are shown as a fold increase obtained by normalization of the geometric MFI value of antibodies to that of control IgG. (E-F) Mouse (E) or human (F) neutrophils were pretreated with or without H₂O₂ (1 µM) or catalase and then incubated with Alexa Fluor 488-conjugated FG in the absence or presence of fMLF. Untreated cells are shown as 100% (white bar). Data represent the mean ± SD (n = 3-4). *P < .05, **P < .01, or ***P < .001 vs WT or vehicle control after analysis of variance (ANOVA) and Dunnett’s test, and #P < .05 or ##P < .01 vs WT or vehicle control after Student t test.