Measurement by MCLA-dependent luminescence method of superoxide formation by N-formylmethionyl-leucyl-phenylalanine-activated inflammatory cells in vitro.
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Activities of human neutrophils and eosinophils to generate O2- were investigated by use of 2-methyl-6-[p-methoxyphenyl]-3, 7-dihydroimidazo [1, 2-α] pyrazin-3-one (MCLA) as a chemiluminescence probe and N-formylmethionyl-leucyl-phenylalanine (fMLP) and cytochalasin B. In some cases, rat mast cells were used instead of human neutrophils and eosinophils. The luminescence intensity was dependent upon the number of neutrophils, and the maximal luminescence intensity was given at 1×10-6M fMLP with a fixed number of the cells. Luminescence intensity caused by the addition of 0.5μM MCLA and 1×10-6M fMLP to a suspension of eosinophils reached maximum at 30s and decreased rapidly thereafter. Biphasic luminescence intensity was observed with 1×10-6M fMLP-stimulated neutrophils and eosinophils in the presence of 5μg/ml cytochalasin B. These MCLA-dependent luminescences by fMLP-stimulated eosinophils were completely abolished by 0.5μM SOD. On the other hand, addition of MCLA and fMLP to a suspension of rat mast cells showed no significant luminescence irrespective of the presence of cytochalasin B.
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