Increased adhesion to blood vessels and TEM of mDia1-deficient neutrophils. (A) In vitro adhesion assay was performed with negatively selected peripheral blood neutrophils on coverslips coated with poly-L-lysine. The relative percentage of adherent mDia1-deficient neutrophils relative to wild-type cells is presented (n = 3 in each group). (B) Negatively selected neutrophils from peripheral blood were seeded on a monolayer of human endothelial cells pretreated with recombinant human tumor necrosis factor (20 ng/mL) for 4 hours. After incubation at 37°C for 45 minutes, the cells were fixed, stained, and counted for 6 random fields (>50 cells in each field). The relative percentage of the transendothelial migrated cells was quantified. (C) Intravital imaging was performed on vasculature of the mDia1 wild-type and knockout mice to evaluate the rolling speed and adhesion of neutrophils in blood vessels. Blood vessels and neutrophils were immunofluorescently stained for PECAM-1 (red, outlined by white dashed lines) and Gr1 (green), respectively. Original magnification ×20. Data were quantified from 6 random fields per group (n = 2 in each group). A representative field is presented. (D-E) Quantitative analysis of the adherent neutrophils and their rolling speed in panel C. (F) mDia1 wild-type and knockout mice were injected twice with anti-CD11b or control antibodies at 50 μg per mouse. The absolute neutrophil count was determined at the indicated time after the second injection (n = 3 mice in each group). IgG, immunoglobulin G; PMN, polymorphonuclear leukocytes.