Figure 5.

(A) Neutrophils were isolated from healthy human peripheral blood and separated using density gradient centrifugation. Purified neutrophils were labeled for multicolor flow cytometry. Expression of DAPI (dead cells), CD45 (general leukocytes), CD16 (neutrophils), CD11b (activated neutrophils), CD62L (primed neutrophils), and CD63 (activated neutrophils) were analyzed and a representative gating strategy was applied to identify activated neutrophils. (B) Quantification of CD11b+ neutrophils. (C) Quantification of CD63+/CD62L– neutrophils. (D) Percentage of activated CD11b+/CD62L–/CD63+ neutrophils in response to fMLF/synthetic ND6 stimulation and/or cyclosporin H (CsH) treatment. (E) Number of migrated neutrophils in response to fMLF/synthetic ND6 stimulation and/or CsH treatment. (F) Extracellular neutrophil ROS production (with HRP to detect extracellular ROS) in response to fMLF/synthetic ND6 stimulation. (G) Extracellular neutrophil ROS production (with HRP to detect extracellular ROS) in response to fMLF/synthetic ND6 stimulation with CsH treatment. (h) Extracellular neutrophil ROS production (with HRP to detect extracellular ROS) in response to fMLF/synthetic ND6 stimulation and CsH treatment (Figure H is Figure F and G combined). Data are means ± standard error (SEM) from n = 3 experiments performed in triplicate. Two-tailed t-test, Mann–Whitney, and 1-way ANOVA with Bonferroni correction and Dunnet’s tests were considered significant if P < .05 with an asterisk (*) indicating P < .05, double asterisks (**) indicating P < .001, and triple asterisks (***) indicating ***P < .0001. ANOVA, analysis of variance; DAPI, 4ʹ,6-diamidino-2-phenylindole; FPR1, formylated peptide receptor-1; HRP, horse-radish peroxide red; IBD, inflammatory bowel disease; UC, ulcerative colitis.