Platelet and neutrophil NOX2 regulate heterotypic neutrophil-platelet interactions during TNF-α-induced venular inflammation. Intravital microscopy of WT and NOX2 KO mice was performed as described in the “Methods” section. Cremaster vascular inflammation was induced by intrascrotal injection of TNF-α. Three hours after TNF-α injection, neutrophils and platelets in inflamed venules were visualized by infusion of Alexa Fluor 647-conjugated anti-Gr-1 and Dylight 488-conjugated anti-CD42c antibodies, respectively, into the mice. (A) Representative images at various time points. Arrows show direction of blood flow. (B-C) Number of rolling and adherent neutrophils. (D-E) The integrated median fluorescence intensities of anti-CD42c antibodies (F platelets) were quantified, normalized by the number of adherent neutrophils and the vessel length, and plotted as a function of time. (E) F platelets in WT and NOX2 KO mice were compared at 60, 120, and 180 seconds after each capture (0 seconds). (F-G) WT and NOX2 KO platelets (F) or neutrophils (G) were isolated and labeled with calcein acetoxymethyl ester (calcein AM). The labeled cells (5 × 10⁷ platelets or 10⁶ neutrophils per mouse) were infused into TNF-α-inflamed mice. Endogenous neutrophils or platelets were visualized by infusion of Alexa Fluor 647-conjugated anti-Gr-1 or Dylight 649-conjugated anti-CD42c antibodies, respectively. Adhesion of the infused platelets to adherent neutrophils (F) and adhesion of endogenous platelets to infused neutrophils (G) were counted and normalized by the number of adherent neutrophils. Data represent the mean ± standard error of the mean (SEM) (n = 22-28 venules in 4 mice per group). *P < .05, **P < .01, or ***P < .001 vs WT control after Mann-Whitney test (E) or vs infusion of WT cells into WT mice (F-G) after Student t test. #P < .05 or ##P < .01 between two groups after Student t test.