Myeloid norbin deficiency increases degranulation, phagocytosis, and NET production. (A) Degranulation. Purified Ncdn^fl/fl and Ncdn^Δmye neutrophils were kept on ice for 45 minutes or were primed with 20 ng/mL TNF-α, 50 ng/mL GM-CSF at 37°C for 45 minutes before stimulation with increasing concentrations of fMLP and 10 μM cytochalasin B (CytB) for 30 minutes at 37°C, as indicated. Gelatinase activity released into the supernatant was analyzed by using in-gel zymography. Left-hand panels indicate representative Coomassie-stained gelatin gels, showing digestion of the gel by gelatinase as white areas. The right-hand panel shows the quantification of gelatinase activity by densitometry, expressed as percentage of primed cells. Data are mean ± standard error of the mean of 7 independent experiments; each dot represents 1 experiment. Statistics comprised two-way analysis of variance with Šidák’s multiple comparisons test. (B) Phagocytosis. Ncdn^fl/fl, LysM^Cre, and Ncdn^Δmye neutrophils were primed with 20 ng/mL TNF-α, 50 ng/mL GM-CSF for 45 minutes at 37°C, plated onto glass coverslips for 30 minutes, and stimulated with zymosan (5 particles/neutrophil) for 30 minutes at 37°C, fixed, stained with FITC-Gr1 antibody and 4′,6-diamidino-2-phenylindole (DAPI), and assessed for the number of zymosan particles within neutrophils by using widefield microscopy and ImageJ analysis. Left-hand panels show representative images of Ncdn^fl/fl cells. The right-hand panel shows the quantification by ImageJ analysis. Data are mean ± standard error of the mean of 3 independent experiments; each dot represents 1 experiment. Statistics comprised one-way analysis of variance with Tukey’s multiple comparisons test. (C) NET production. Purified Ncdn^fl/fl and Ncdn^Δmye neutrophils were seeded onto glass slides and allowed to adhere for 30 minutes at 37°C before being stimulated with serum-opsonized S aureus (10 bacteria per neutrophil; filled symbols), or mock-stimulated (open symbols), for the indicated periods of time. Sytox Green dye was added to samples 15 minutes before their time point, samples were live imaged, and NETs quantified by using ImageJ software. Phase contrast was used to count total cells. Left-hand panels show representative images. The red arrow denotes a dead cell, the white arrow a NET. The right-hand panel shows the quantification according to ImageJ analysis. Data are mean ± standard error of the mean of 3 independent experiments. Statistics comprised two-way analysis of variance with Šidák’s multiple comparisons test.