Measurement of superoxide anion production using maximal rate of cytochrome (III) C reduction in phorbol ester stimulated neutrophils, immobilised to microtiter plates.

In the present investigation, a method of studying the maximal rate of superoxide anion (O2.-) production in immobilised human neutrophils using a microtiter plate technique has been developed. The rate of O2.- production was determined from the rate of reduction of cytochrome (III) C, studied as the increase in absorbance at 550 nm. The protein kinase C activator, phorbol 12-myristate 13-acetate, was used to stimulate O2.- production. Neutrophils were evenly immobilised as a monolayer to microtiter culture plates to provide a reproducible exposure to the medium. Phorbol ester stimulated O2.- production was inhibited by staurosporine, a well-known inhibitor of protein kinase C, and by diphenylene iodonium, a potent NADPH-oxidase inhibitor, with IC50-values in this assay of 20 and 220 nm, respectively. The extracellularly produced O2.- was removed by superoxide dismutase with a half maximal effect of 0.6 microgram/mL. The maximal production rate of O2.- could therefore be estimated by addition of 20 micrograms/mL superoxide dismutase. Several antioxidants, including butylated hydroxytoluene, nordihydroguairetic acid, probucol and alpha-tocopherol, were studied and showed neither an effect on O2.- production nor a scavenging effect. This new method was highly reproducible, and the continuous measurement of O2.- production was very useful for validating the effect of inhibitors. The developed microtiter technique using immobilised cells has a large capacity and allows different compounds to be tested under comparable conditions, since they are exposed to the cells in a similar way. This is also the first test model which describes O2.- production as the maximal rate of cytochrome (III) C reduction.