Abstract
Luminol-, isoluminol- or lucigenin-enhanced chemiluminescence (CL) was used to measure the production of reactive oxygen species by rat blood leukocytes. Opsonized zymosan (OZ), phorbol-12-myristate-13-acetate (PMA), calcium ionophore A23187 (Ca-I) or N-formyl-Met-Leu-Phe (fMLP) were used as activators. The CL signal of isolated blood leukocytes decreased in rank order of luminol > isoluminol > lucigenin. The kinetic profiles of luminol- and isoluminol-enhanced CL were similar upon stimulation by each activator tested. The remarkably higher luminol and isoluminol CL responses were obtained after OZ stimulation when compared with other activators. However, when lucigenin was used, the PMA- and OZ-stimulated CL were comparable. The presence of plasma increased OZ-activated CL because of the enhanced phagocytosis of OZ. This was demonstrated by determining the phagocytosis of the fluorescent OZ using a flow cytometer. In contrast, the presence of plasma decreased PMA-activated CL, due to the antioxidant properties of plasma as determined by the CL method. As far as whole blood is concerned, only OZ activated luminol-enhanced CL was reliable. Blood volumes over 5 microL decreased CL activity due to the scavenging ability of erythrocytes. The results suggest that 0.5 microL whole blood is sufficient for routine luminol-enhanced CL analysis of whole blood oxidative burst in rats.
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