Fig. 1. 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR) staining of macrophages and neutrophils is retained in vivo and does not interfere with adhesion to or transmigration across a monolayer of immortalized murine endothelial cells (fEnd5) in vitro . DiR-labeled and control leukocytes were tested on tumor necrosis factor-α (TNFα)–stimulated and control endothelial cells. (A ) No differences in the number of adhering macrophages per high-power field (hpf) were detected in DiR-labeled and control macrophages on resting or activated endothelial cells (n = 6, P = not significant [n.s.]). (B ) Double-labeled leukocytes were allowed to transmigrate across an endothelial cell monolayer grown on transwell inserts. DiR labeling did not affect macrophage transmigration across resting or tumor necrosis factor-α stimulated endothelial cells (n = 6, P = not significant). Similar results were obtained for DiR-labeled neutrophils. (C ) The dye did not affect adhesion (n = 6, P = not significant) and no significant differences were seen for (D ) neutrophil transmigration (n = 6, P = not significant). (E ) Oxidative burst was detected in neutrophils by dichlorofluorescin diacetate staining and was measured in phorbol 12-myristate 13-acetate (PMA)–activated and control neutrophils after 160 min of stimulation. The mean fluorescence intensity (MFI) of dichlorofluorescein diacetate resulting from neutrophil-generated oxidative burst did not differ in DiR-labeled and control cells. AU = arbitrary units. (n = 4, P = not significant.) (F ) The staining procedure was highly efficient. Double label of enhanced green fluorescent protein (eGFP) and DiR was detected in more than 97% of the cells before injection. Double-labeled macrophages were detected in the lavage at day 3. (G ) Only a minor fading of DiR fluorescent intensity was observed when cells were recovered from two lavages at day 3 and compared with cells before injection by fluorescence microscopy (n = 4, P < 0.05).