Blood was drawn into a heparinized syringe from healthy human volunteers and leukocytes were isolated by sedimentation in the presence of 6% dextran. After then, neutrophils and eosinophils were purifid on a Conray-Ficoll and Percoll gradient respectively. Rat serosal mast cells were also purified on a Percoll gradient. The cells were incubated with N-formyl-methionyl-leucyl-phenylala-nine (fMLP) and O-2 generation was measured by 2-methyl-6- [p-methoxyphenyl] -3, 7-dihydroimidazo [1, 2-a] pyrazin-3-one (MCLA) -dependent luminescence.
Addition of 0.5μM MCLA and 10-6 M fMLP to a suspension of neutrophils caused a marked luminescence within 1 min, which was inhibited by 0.5μM superoxide dismutase (SOD) . The luminescence intensity was dependent upon the number of neutrophils and the maximal luminescence intensity was given at 10-6 M fMLP with a fixed numbers of the cells (1×105) .
Biphasic luminescence light intensities were observed by 10-6 M fMLP-stimulated neutrophils and eosinophils in the presence of 5μg/ml cytochalasin B and either of them was inhibited by 0.5μM SOD.
On the other hand, addition of 0.5μM MCLA and 10-6 M fMLP to a suspension of rat serosal mast cells showed no significant luminescence even in the presence of cytochalasin B, suggesting the absence of fMLP receptor on the mast cell membranes.
