Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader

Two simple semiautomated microassays for the measurement of superoxide (O-2) and hydrogen peroxide (H2O2) production by cultured macrophages (MPs) are described. The measurement of O-2 is based on the reduction of ferricytochrome c as assayed by the increase in its absorbance at 550 nm. Quantitation of H2O2 is based on the horseradish peroxidase (HRPO)-dependent oxidation of phenol red which is assayed by its increased absorbance at 600 nm. MPs are cultured in monolayers in 96-well flat-bottom tissue culture plates and covered with 100 mul amounts per well of either a ferricytochrome c solution containing phenol red and HRPO. Following the addition of an agent eliciting an oxidative burst (OB) and incubation of the plates at 37 degrees C for various time intervals, the changes in the absorbance of ferricytochrome c and phenol red, respectively, are measured directly in the wells of the tissue culture plates with the cells in situ, by using an automatic 8-channel photometer which reads absorbances vertically through individual wells. This instrument, which was originally designed for reading enzyme immunoassays in microtitration plates, can be easily adapted for use in the above test, when fitted with interference filters with wave lengths of 550 nm (for the assay of O-2) and 600 nm (for the assay of H2O2). The principal advantages of this techniques are: the ability to perform the assays directly in the culture plates with cells in situ; the small amounts of cells and reagents needed; its sensitivity and reproducibility; the ease with which kinetic experiments can be done; the large number of samples which can be tested in parallel, and especially the speed and convenience offered by the automated reading and printout of absorbance values.