A sensitive detection of neutrophil activation by fluorescence quenching of membrane inserted singlet oxygen probe

Fluorescence emission intensity of 1,3-diphenylisobenzofuran incorporated in polymorphonuclear granulocytes plasma membranes was investigated in basal conditions and during stimulation with different activators. Phorbol myristate acetate, known as the most effective "oxygen burst" inducer, produced a larger decrease in 1,3-diphenylisobenzofuran fluorescence intensity than 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) and N-formyl-methionyl-leucyl-phenylalanine, known as weak stimulants of oxygen uptake. Diphenyl iodonium an inhibitor of leukocyte NADPH oxidase, and the singlet oxygen selective trap alpha-terpinene inhibited the quenching effect of phorbol myristate acetate on 1,3-diphenylisobenzofuran fluorescence. These data suggest formation of singlet oxygen in activated leukocytes and demonstrate that measurement of 1,3-diphenylisobenzofuran fluorescence intensity provides a new sensitive method of detection of neutrophils activation.