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Luminol-enhanced Assay for Superoxide Anion (O2 face="Symbol" ,,,)

but also requires a large number of cells (106) per assay. The third method, luminol-mediated chemiluminescence, is the most sensitive method of all; accurate readings can extend three orders of magnitude over the signal range.

The Luminol Chemiluminescence Reaction

The chain of events for the luminol chemiluminescence (CL) assay is believed to involve cell surface receptors, such as those binding to complex immunoglobulin G (Fc receptors) or bacterial formylated peptides (f-met-leu-phe receptors), which are stimulated by their specific agonists. The activated receptor triggers the production or activation of NADPH oxidase, leading to the production of superoxide anion. Superoxide anion is subsequently released into the extracellular environment where it can oxidize luminol and available lipids and proteins. This oxidation results in chemiluminescence, a release of photons (light), which can be measured by a luminometer. Figure 1 illustrates Stratagenes enhanced chemiluminescent procedure for measuring superoxide anion.

When concentrations of superoxide anion are very low, assay sensitivity may need to be enhanced. Some enhancement reagents, such as iodophenol, are phenolic-based compounds. Unfortunately, such enhancers are toxic to live cells and denaturing to some components of subcellular systems; hence, they cannot be used for in situ assays. However, Stratagenes specific enhancement reagent increases photon output but is noncytotoxic and does not denature cellular components used in assaying live cell activity.

Assay for Superoxide Anions in U-937 cells

Cell cultures were provided with an analyte suspected of being able to generate superoxide anion; luminol and enhancement reagents were then added. Superoxide anion generated was measured as a result of luminol oxidation.

As a model, U-937 cells (monocytic-like, histocytic human lymphoma) were cultured in RPMI supplemen
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