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

Non-cytotoxic chemiluminescent enhancer for increased sensitivity

Luminol-enhanced Assay for Superoxide Anion (O₂ )

Danny Q. Hoang
Stratagene

Lorraine C. Pfefferkorn
Dartmouth School of Medicine, Dept. of Microbiology, Hanover, NH

Stratagene introduces the LumiMax detection assay, a simple and unique chemiluminescent-based method to qualitatively detect the presence of superoxide anions in cell culture. In addition, the kit provides a specific enhancement reagent to amplify chemiluminescent signal output that is noncytotoxic and nondenaturing to live cells. Superoxide anion (O₂ ), a potent oxidant, is a short-lived oxygen radical that is released into the extracellular environment of stimulated leukocytes (monocytes, macrophages, and polymorphonuclear leukocytes).¹ Superoxide anion is synthesized when NADPH-oxidase, a plasma membrane enzyme complex containing cytochrome b 558, transfers an electron to molecular oxygen. Superoxide anions are implicated in several key cellular processes, including oxidative stress damage,² tumor promotion,³ and, most recently, cell growth and DNA synthesis via their involvement in the cell signaling pathway of proto-oncogene ras.⁴

At present, superoxide may be detected by measuring the reduction of exogenously supplied cytochrome c,⁵ the uptake of oxygen from the medium (measured by the Clark electrode⁶), or luminol-mediated chemiluminescence.⁷ The first method has limited sensitivity and is complicated by the reoxidation of superoxide-reduced cytochrome c by contaminants and cell lysis. The oxygen-uptake method is not only less sensitive than cytochrome c reduction
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