COMPARATIVE USE OF 2,7-DICHLOROFLUORESCIN DIACETATE, DIHYDRORHODAMINE-123, AND HYDROETHIDINE TO STUDY OXIDATIVE-METABOLISM IN PHAGOCYTIC-CELLS

Tests carried out with model systems to assess the sensitivity of 2,7-dichlorofluorescin diacetate, dihydrorhodamine 123, and hydroethidine to the reactive oxygen species generated in these systems showed that ail three fluorochromes are sensitive to hydroxyl and superoxide radicals and to hypochlorite ions and less sensitive to hydrogen peroxide. They do not react with singlet oxygen. Hydroethidine reacts with superoxide radicals in the 1:1 molar ratio. The relative proportions of reactive oxygen species generated extra- and intracellularly were estimated for phorbol myristate acetate-stimulated peritoneal and alveolar rat macrophages and for polymorphonuclear leukocytes from the blood of healthy donors. In activated leukocytes of different types, intracellular generation accounted for approximately 20% of the total reactive oxygen species produced. Intracellular production of these species was found to show considerable cyanide sensitivity, which indicates that cyanide-resitant NADPH oxidase located in the plasma membrane is not implicated in the generation of reactive oxygen species.