Microassays for superoxide and hydrogen peroxide production and nitroblue tetrazolium reduction using an enzyme immunoassay microplate reader

This chapter describes three assays for the quantitation of the cyanide resistant oxidative metabolism of macrophages. Macrophages of various tissue origins produce copious amounts of superoxide (O₂^–) and hydrogen peroxide (H₂O₂) when adequately stimulated. This process is accompanied by a marked increment in oxygen uptake and an increased utilization of glucose via the hexose monophosphate shunt (HMPS). The coordinated sequence of reactions is known as the “oxidative” or “respiratory” burst. The three methods have in common the measurement of O₂^– and H₂O₂ production and nitroblue tetrazolium (NBT) reduction by cells cultured in 96-well microplates with the aid of an enzyme immunoassay microplate reader fitted with appropriate filters for the photometric determination of the respective reaction products. The microassay of O₂^– production is based on the reduction of ferricytochrome c by O₂^–, the specificity of reduction being controlled by its inhibition by superoxide dismutase. The length of time for which O₂^–production occurs at a linear rate depends on the type of macrophage, on the cell density, and on the nature of the stimulus.