CARBOXYFLUORESCEIN DIACETATE LABELING DOES NOT AFFECT ADHESION MOLECULE EXPRESSION OR FUNCTION IN HUMAN NEUTROPHILS OR EOSINOPHILS

Fluorescently labeled leukocytes are commonly used in in vitro and in vivo experimental systems. However, the effects of fluorescent labelin g on the expression and function of leukocyte adhesion molecules has n ot been examined in part because the extreme intensity of fluorescence tends to obscure signals from other fluorochromes used for dual color analysis. We have utilized a novel technique involving a 7-amino-4-me thylcoumarin-3-acetic acid (AMCA) fluorophore-conjugated F(ab')(2) fra gment excitable in the ultraviolet wavelength range (350-450 nm) and d ual-laser flow cytometry to determine if labeling of human neutrophils and eosinophils with the fluorescent dye 5-(6)-carboxyfluorescein dia cetate (CFDA) alters surface expression of the primary leukocyte adhes ion molecules involved in leukocyte-endothelial interactions. Simultan eously, adhesion molecule function was assessed by comparing the abili ty of CFDA-labeled vs. control cells to adhere to cultured human umbil ical vein endothelial cells (HUVEC) and purified immobilized adhesion molecules. Isolated human eosinophils and neutrophils were fluorescent ly labeled by incubation with CFDA. Flow cytometric comparisons of lab eled and unlabeled cells demonstrated that fluorescence labeling of ne utrophils and eosinophils with CFDA did not alter basal surface expres sion of the beta(2) integrins (i.e., CD11a CD11b or, CD18). Stimulatio n of neutrophils with fMLP and eosinophils with PMA resulted in increa sed surface expression of CD11b and CD18 which was not altered by CFDA labeling. Likewise, CFDA labeling of neutrophils and eosinophils did not significantly alter their integrin-dependent adhesion to activated HUVEC under static or rotational conditions. Similarly, adhesion to i mmobilized recombinant E- and P-selectin was unaltered. These data dem onstrate that fluorescent labeling of human neutrophils and eosinophil s with CFDA does not alter surface expression or function of several a dhesion molecules necessary for leukocyte-endothelial interactions. Th e use of CFDA-labeIed cells in experiments employing intravital micros copy should therefore provide valid information on adhesion molecule f unction in vivo.