Norbin-deficient neutrophils have increased cell surface levels of the GPCRs C5aR1 and CXCR4 but normal surface levels of other receptors and adhesion molecules. (A) Ncdn^fl/fl and Ncdn^Δmye bone marrow cells were kept on ice (basal; open symbols) or were primed with 20 ng/mL TNF-α, 50 ng/mL GM-CSF for 45 minutes at 37°C before transfer onto ice (filled symbols). Cells were stained to identify neutrophils and quantify the mean levels of C5aR1, CXCR4, CXCR1, and CXCR2 on the neutrophil surface by flow cytometry. (Top, middle panel) Bone marrow cells were incubated for 30 minutes at 37°C without or with 100 nM C5a, or C5a was added for the last 10 minutes as indicated, before transfer onto ice and quantification of the mean C5aR1 level on the neutrophil surface. Data are mean ± standard error of the mean (SEM) of 3 independent experiments; each dot is the mean of 1 experiment. (B) Ncdn^fl/fl, LysM^Cre, and Ncdn^Δmye bone marrow cells were treated as in panel A and stained to identify neutrophils and quantify the mean levels of L-selectin, PSGL-1, LFA-1, and Mac-1 on the neutrophil surface by flow cytometry. Data are mean ± SEM of 3 to 8 independent experiments; each dot is the mean of 1 experiment. (C) Integrin avidity. Purified Ncdn^fl/fl and Ncdn^Δmye neutrophils were primed with 20 ng/mL TNF-α, 50 ng/mL GM-CSF for 45 minutes at 37°C before incubation with AF594-labeled anti-CD18 antibody for 3 minutes at 37°C in the presence (filled symbols) or absence (open symbols) of 1.5 μM fMLP. Cells were fixed and allowed to settle onto electrostatically charged slides before imaging and image analysis for the presence and localization of integrin clusters. A total of 30 to 40 cells were analyzed/condition/experiment. Data are mean ± SEM of 3 independent experiments; each dot represents 1 experiment. Statistics in panels A to C comprised two-way analysis of variance with Šidák’s multiple comparisons test.