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.
Björquist
P
P
Department of Biochemistry, ASTRA HASSLE AB, Mölndal, Sweden.
Palmer
M
M
Ek
B
B
D000975:Antioxidants / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000378:metabolism*; D006801:Humans; D018375:Neutrophil Activation; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D010084:Oxidation-Reduction; D013481:Superoxides / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D000975:Antioxidants; D003574:Cytochrome c Group; D013481:Superoxides; D013755:Tetradecanoylphorbol Acetate