59] Kinetic microplate assay for superoxide production by neutrophils and other phagocytic cells

Abstract

This chapter presents a kinetic microplate assay for superoxide production by neutrophils and other phagocytic cells. Superoxide production by phagocytic cells is usually monitored by either continuous or end-point assays in which cytochrome c reduction is measured with single- or dual-beam spectrophotometers. The continuous assay is the preferred method as the generation of O2– by phagocytes is not linear with respect to time. Human neutrophils of over 98% purity are prepared using acid–citrate–dextrose as the anticoagulant, dextran to sediment erythrocytes, and Ficoll–Hypaque density gradient centrifugation to separate mononuclear cells from neutrophils. Neutrophils are suspended in phosphate-buffered saline (PBS). Monocytes or macrophages can be substituted for neutrophils in the microplate O2– assay. To facilitate the processing and calculation of the rates of O2– production, the kinetic software designed for the Vmax, SOFTmax Version 2.0 (Molecular Devices), is used. This software is capable of determining the maximal rate of absorbance change by calculating the first derivative of the absorbance time course in each well. This feature is particularly useful for stimuli such as phorbol 12-myristate 13-acetate (PMA), which exhibits an approximately 45 second lag before O2– is generated at a maximal rate. This feature is also convenient for the N-formyl-L-methionyl-L-leucyl-L-phenylalanin (FMLP) experiments as the rate of O2– production with this stimulus decreases rapidly after 2 min.