Adaption of a Colorimetric Assay for Hydrogen Peroxide Released by Activated Macrophages
Malleck, Marjorie A.
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Macrophages are secretory and phagocytic cells which acquire heightened metabolic, microbicidal, and cytocidal activity when activated. Activated macrophages exhibit increased oxygen uptake and secretion of toxic oxygen intermediates such as 0; and H202• Hydrogen peroxide released by macrophages has been implicated in the killing of parasites and tumor cells. An assay was developed by Edgar pick to detect the amount of H20 2 released by macrophages in a monolayer based on the horseradish peroxidase (HRPO)mediated oxidation of phenol red by H202• The assay was then adapted by Pick for use with an automatic a-channel spectrophotometer (Multiskan). This project adapted these techniques for use at Upjohn. The optimum parameters for this assay with mouse macrophages were determined, including cell density, and incubation times with the stimulants of H202 release tested. A chemical stimulant, phorbal myristate acetate, caused the macrophages to release large amounts of H202 , while the physiologic stimulant, macrophage activating factor, derived from mitogen stimulated spleen cells, did not. Five murine macrophage cell lines, stimulated by phorbal myristate acetate, failed to release H202 by this method. This assay could be used in the future for the screening of potential anti-inflammatory, anti-microbial, or anti-cancer compounds for capacity to stimulate macrophages to release H202•