Isoluminol-enhanced chemiluminescence: A sensitive method to study the release of superoxide anion from human neutrophils

doi:10.1016/0891-5849(95)02189-2

Free Radical Biology and Medicine

Volume 20, Issue 6, 1996, Pages 785-792

https://doi.org/10.1016/0891-5849(95)02189-2 Get rights and content

Abstract

A very sensitive chemiluminescence (CL) method was developed for the determination of respiratory burst products generated by the NADPH-oxidase in human neutrophils. Despite the fact that the CL reaction is peroxidase dependent, hydrogen peroxide was found not to participate in the light generating reaction. Phagocytic cells were mixed with isoluminol, a chemiluminescence substrate that detects extracellularly released oxygen species only. Owing to the fact that the availability of released cellular myeloperoxidase is a limiting factor in the reaction, extra peroxidase (horseradish peroxidase; HRP) has to be added to the measuring system. From the fact that the response to phorbol myristate acetate (PMA), as well as to formyl-methionyl-leucyl-phenylalanine (fMLP) was almost totally inhibited by superoxide dismutase (SOD), we conclude that O₂^·− is the reacting oxygen species measured. The assay system described is well suited for real-time studies of superoxide anion release from activated neutrophils. With the technique, the release of O₂^·− can be detected from as few as 250 neutrophils.

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Original contributionIsoluminol-enhanced chemiluminescence: A sensitive method to study the release of superoxide anion from human neutrophils

Abstract

Tissue destruction by neutrophils

N. Engl. J. Med.

Dual granule localization of the dormant NADPH oxidase and cytochrome b559 in human neutrophils

Eur. J. Haematol.

Ultrastructural lokalization of cytochrome b in the membranes of resting and phagocytosing human granulocytes

J. Clin. Invest.

The inflammatory reaction

The electron transport chain of the microbicidal oxidase of phagocytic cells and its involvement in the pathology of chronic granulomatous disease

J. Clin. Invest.

The genetic basis of chronic granulomatous disease

Immunol. Rev.

Laboratory manual of neutrophil function

Superoxide generation by digitonin-stimulated guinea pig granulocytes

A quantitative fluorimetric assay for the detection of oxidant production by polymorphonuclear leukocytes: Its use in the simultaneous fluorimetric assay of cellular activation processes

Anal. Biochem.