Neutrophil migration requires IL-6 signaling. Neutrophils were preincubated with PBS (control) or anti-IL-6R blocking antibody for 30 minutes before starting the migration experiment, and the experiment was run in the continued presence of the antibody. (A) Representative images of neutrophil migration. Neutrophils stained with calcein AM. Bacterial gradient direction shown in red. White line indicates the edge of the endothelial lumen. Scale bar, 100 µm. (B) The number of neutrophils outside the lumen was counted at 1-hour intervals, using particle analysis in FIJI. (C) The distance from the lumen edge was measured for all in-focus neutrophils outside the lumen at 4-hour intervals. Each bar represents the mean plus SEM. (D) The number of migrated neutrophils (% maximum migration) in each region was determined at 4-hour intervals. (E) The number of PI-positive cells in the whole image was counted using FIJI and normalized to the number of calcein AM-positive cells at t = 0 hours. Bars represent the mean plus SEM. Data quantified from 11 lumens (Control) or 14 lumens (anti-IL-6R) across 4 independent experiments. Asterisks represents significance between conditions at each point. *P < .05; **P < .01; ***P < .001; and ****P < .0001.