Isolation and Functional Analysis of Neutrophils

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Abstract
This unit describes the isolation of high-purity neutrophils (or polymorphonuclear leukocytes; PMN) and the assays that can be performed to assess their function. An assay for the in vitro study of phagocytic capacity is presented, and microbicidal assays using lysostaphin and differential centrifugation are also described. During phagocytosis or on exposure to soluble agonists, neutrophils exhibit a marked increase in oxidative metabolism which is mediated by activation of NADPH oxidase and results in the formation of superoxide anion as well as hydrogen peroxide and other reactive species. The nitroblue tetrazolium reduction slide test is presented, and is a nonquantitative screening test for the presence of reactive oxygen products. The quantitation of superoxide by the kinetic and static assays are detailed, and a “broken cell” assay is also outlined.Finally, measurement of hydrogen peroxide formation is described.

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