Migration to P aeruginosa requires an endothelium. Neutrophil migration was measured across an unlined lumen (No Endothelium) or an endothelial-lined lumen (Endothelium) to a source of P aeruginosa. (A) Representative images of neutrophil migration. Neutrophils stained with calcein AM. Bacterial gradient direction shown in red. White line indicates the lumen edge. 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-E) The percentage of migrated neutrophils relative to the maximum number of migrated neutrophils at any point per condition was determined in four, 100-µm regions at increasing distance from the lumen (D) and plotted as an area graph (E). (F) Statistical significance for migrated neutrophils (% maximum migration) in each region was determined at 4-hour intervals. Bars represent the mean plus SEM. All data were quantified from 13 lumens (No Endothelium) or 14 lumens (Endothelium) across 5 independent experiments. Asterisks represents significance between conditions at each point. *P < .05; **P < .01; ***P < .001; and ****P < .0001.