Characterization of bone marrow–derived macrophages (BMMs). (A) BMMs obtained by culture of bone marrow cells from 7- to 10-week-old wild-type, TF^ΔCT, PAR2^−/−, or TF^ΔCT/PAR2^−/− mice were analyzed by flow cytometry for the macrophage surface markers F4/80 and CD11b. (B) Expression of costimulatory molecules on BMMs after overnight stimulation with LPS (1 μg/mL). Isotype-matched control antibody staining (dotted histogram) or specific staining of non–LPS-stimulated (shaded area) or LPS-stimulated (solid line) BMMs are shown; an example of at least 3 independent experiments is given. (C) Morphology of wild-type and TF^ΔCT BMMs with and without overnight LPS stimulation by phase-contrast microscopy (upper panel) or after phalloidin staining (red) by confocal microscopy; nucleus is stained with ToPro3 (blue) (lower panel).[AU34} (D-F) Effector functions of wild-type or TF^ΔCT BMMs. Overnight LPS (1 μg/mL) stimulation leads to similar up-regulation of NOS2 by Western blotting (D) and nitric oxide (NO) production (E) determined using the Griess reagent³⁷  (n = 3). (F) FcγR-mediated phagocytosis of IgG-opsonized sheep red blood cells by wild-type and TF^ΔCT BMMs.