A method for isolation and fluorescent labeling of rat neutrophils for intravital microvascular studies

Abstract

A method for the isolation, fluorescent labeling, and reinfusion of rat neutrophils for in vivo investigation of white blood cell function in the microcirculation is described. The cell surface morphology and function of the labeled and unlabeled neutrophils were compared in vitro and in vivo. The morphology of the labeled and unlabeled cells was visually assessed by differential interference contrast microscopy before and after exposure to the chemotactic peptide FMLP. Labeled and unlabeled cells were morphologically similar under normal and stimulated conditions. The chemotactic responsiveness of the labeled and unlabeled cells was evaluated in vitro by quantifying the movement of cells across a porous membrane in response to FMLP. No significant difference in chemotactic responsiveness was found. The in vivo behavior of the labeled neutrophils was quantitatively studied by examining their interactions with the venular endothelium in the rat cremaster muscle. Rolling velocity for labeled neutrophils and unlabeled white blood cells was measured at different flow velocities and wall shear rates. No difference was found between the labeled and unlabeled cells. It is concluded that this isolation and labeling procedure yields cells possessing normal morphology and function. These labeled neutrophils may be useful for in vivo study of their behavior in the microcirculation.