An endothelial lumen increases neutrophil lifetime. Propidium iodide staining was used to monitor neutrophil lifetime in unlined and endothelial-lined lumens. (A) Calcein AM was used to stain neutrophils (green), and PI was used to stain dead cells (magenta). Phase images (left) are shown for lumen edge visualization. The gradient direction is indicated in red. Images are representative of 4 independent experiments. Scale bar, 100 µm. (B) 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. (C) 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 mean plus SEM. All data were quantified from 12 lumens (No Endothelium) or 11 lumens (Endothelium) across 4 independent experiments, and from 9 lumens (Control) or 8 lumens (anti-GM-CSF) across 3 independent experiments. Asterisks represent significance between conditions at each point. *P < .05; **P < .01; ***P < .001; and ****P < .0001.