Engagement of FcγRs results in the formation of neutrophil extracellular traps in vivo. Requirement for FcγRIIA but not the NADPH oxidase, myeloperoxidase (mpo), or neutrophil elastase. The RPA was induced in the cremaster of mice. After 3 hours, the cremaster muscle was exteriorized for intravital microscopy, and the mice were given an intravenous injection of Sytox Green, a DNA binding dye, and tetramethylrhodamine (TRITC) conjugated Dextran (70 kDa), which delineates blood vessels. (A) Left panel: NET-like structures (green) were observed in wild-type (WT) mice subjected to the RPA (BSA/anti-BSA) but not in mice given BSA alone that localized primarily to the extravascular space. Scale bar represents 50 μm. Middle and right panels: The image (BSA/anti-BSA) was subjected to surface analysis of blood vessels (red) and extravascular (blue) and intravascular (green) NET structures. The analysis confirmed the presence of NET-like structures mostly in the extravascular space with a very small fraction present within blood vessels (arrow). (B) Top panels: NET-like structures (arrow) were visible in the cremaster muscle of WT mice (WT, −), although they were absent in WT mice treated with neutrophil-depleting Gr-1 antibody (WT, PMN-depleted). NET-like structures were visible in gp91^−/− mice 3 hours after RPA. Scale bar represents 50 μm. Bottom left panel: A graph of the average ± SEM of the number of NETs per millimeter squared of cremaster tissue (n/mm²) for each genotype/condition is shown. N = 3 independent experiments. ***P < .001. n.s. indicates not significant. Bottom right panel: Neutrophils isolated from wild-type and gp91^−/− mice were treated with PMA, and ROS generation was evaluated in real-time using a luminol-based assay. (C) Top panels: NET-like structures were visible in the cremaster muscle of WT, mpo^−/−, and elastase ^−/− mice subjected to the RPA. Bottom panel: The quantification of NETs was conducted as described in panel B. N = 3 or 4 mice per genotype. (D) NETosis was evaluated in FcγRIIA/γ^−/−, FcγRIIIB/γ^−/−, γ^−/− mice, and FcγRIIA/γ^−/−/Mac-1^−/− mice subjected to the RPA. Left panels: NET-like structures were present in the cremaster muscle of FcγRIIA/γ^−/−, but not FcγRIIIB/γ^−/− or γ^−/− mice. In FcγRIIIB/γ^−/− and γ^−/− mice, Sytox Green was present within cells, which probably reflect the presence of permeable neutrophils. Scale bar represents 50 μm. Right panels: The quantification of NETs for indicated animals was conducted as described in panel B. n = 3 mice per group. ***P < .001. (B-D) Signals coming from Sytox Green-positive intact cells (arrowhead), excluded from this analysis, were similar in all groups.