In vivo labeling of neutrophils using a fluorescent cell linker

To study neutrophil circulation time and trafficking in vivo requires labeling the cells so that their movement can be followed temporally and spatially. Labeling procedures used to date, however, have relied on ex vivo separation and labeling methods, an undesired consequence of which may be neutrophil activation. Moreover, the labeled cells preferentially stick in the pulmonary circulation for several hours upon reinjection into the host. Therefore, we devised an alternate labeling procedure that relied on intra-arterial infusion of the fluorescent phagocytic cell linker PKH26. We infused PKH26 (5.5 x 10(-6) M for 1 h) into six awake sheep and measured systemic and pulmonary hemodynamics and lung lymph dynamics continuously for 3-4 h after the infusion. We collected simultaneous arterial and venous blood samples hourly for 4 h, and again 24 h after the infusion ended, for white blood cell and neutrophil differential counts and to identify labeled cells in fresh smears by fluorescence and differential interference contrast (DIC) microscopy. Infusion of PKH26 had minimal and transient physiologic effects on systemic and pulmonary artery pressure, lung lymph flow, and leukocyte counts. Labeled cells were present in venous blood after the infusion for at least 24 h. DIC microscopy observation of the blood smears indicated that the fluorescently labeled cells were exclusively neutrophils. Unstimulated superoxide anion release from neutrophils isolated 2 and 24 h after PKH26 infusion was not different from baseline release. Phorbol myristate acetate-stimulated release was not affected either. The labeled neutrophils responded to chemoattractants by migrating to extravascular sites. Our results indicate that neutrophils can be selectively labeled in vivo, after which trafficking of the labeled neutrophils can be determined.