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

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Abstract

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 O2production occurs at a linear rate depends on the type of macrophage, on the cell density, and on the nature of the stimulus.

Original languageEnglish
Pages (from-to)407-421
Number of pages15
JournalMethods in Enzymology
Volume132
Issue numberC
DOIs
StatePublished - 1 Jan 1986

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