Comparison of four methods for the isolation of murine blood neutrophils with respect to the release of reactive oxygen and nitrogen species and the expression of immunological receptors

Abstract

Neutrophils are phagocytic leukocytes that represent one of the first lines of defense during infection. These cells are phagocytose and kills microbes through the production of toxins such as superoxide anions, hydrogen peroxide, and nitric oxide. The isolation of neutrophils from either human beings or animals, such as mice, is a common first step for researchers when investigating innate immunity to microbes. Fortunately, there are literature reports that compares the separation properties and functional responses of cells isolated from different animal species. However, researchers still encounter technical problems with regard to purification, viability, and recovery. In this study, we describe different methods for the isolation of neutrophils from mouse blood. We compared the purity, viability, yield, and the expression of immunological receptors and functional responses such as ROS production induced by opsonized particles of zymosan and phorbol 12-myristate 13-acetate-stimulated of neutrophils isolated in four procedures. In this comparative study, we observed that Percoll was most appropriate for the isolation of mouse neutrophils, because we obtained the most pure and viable cell preparation through this process. Neutrophils isolated with a Percoll gradient demonstrated a greater production of reactive oxygen and nitrogen species compared to the other techniques. Additionally, we observed a slight increase in the expression of CD64 when cells were purified with the gelatin method and over 95 % expression of the CD16 and CD32 receptors with all methods evaluated.