37] Activation of macrophages with oxidative enzymes

Abstract

This chapter describes two assay methods used for activating macrophages with peroxidases. The first one employs a target cell cytostasis assay, and the second determines the increase in the “oxidative burst” as measured by the increase in superoxide production, using the chemiluminescence of luminol as the indicator. The microassay for target cell toxicity is different from other cytotoxicity assays as it does not require the presence of a radioactive marker. A fixed number of target cells are exposed to activated macrophages for a period of 48 hours; and at the end of this time, the cells are stained with a dye. The amount of stain present in wells containing target cells and activated macrophages is then compared with the amount of stain present in wells containing target cells with nonactivated macrophages. One of the functions of phagocytic cells is to ingest and kill microorganisms. These cells emit light while ingesting particles. Light arises from the production of high-energy compounds produced during bactericidal activity. This spontaneous luminescence can be used as an assay of phagocytic function or as a sensitive means to quantitate microbicidal metabolic activity.