DOC 412-012

Detection of superoxide generation from cultured vascular cells using the Hidex Chameleon II, a high sensitivity multi-technology microplate reader
Dr Richard Siow and Prof Giovanni Mann, Cardiovascular Division, Kings College, University of London, UK
Superoxide (Lucigenin MLU)

INTRODUCTION
Reactive oxygen species play a critical role in the pathogenesis of many diseases. The superoxide anion (O2-) is among the most important of these, not only because of its rapid reactions but also because it initiates the production of other reactive oxygen species. Chemiluminescence is frequently used to detect O2- in neutrophils and vascular cells. On exposure to O2-, chemiluminescent probes release a photon, which in turn can be detected by a scintillation counter or a luminometer. Because most of these compounds are cell permeable, the O2- measured reflects extracellular as well as intracellular O2- production. Among the chemiluminescent compounds used, bis-N-methylacridinium nitrate (lucigenin) remains the most widely used. Reactions involved in lucigenin chemiluminescence are as follows:
1) O2-+LC2+ LC++O2; 2) LC++O2LCO2; 3) LCO2 2N-methylacridone+h

RESULTS
Fig 1 Assessment of superoxide generated by xanthine oxidase (XO) in a cell free system using hypoxanthine as substrate showing that the Hidex Chameleon II plate reader was able to reproducibly detect O2levels generated from 100 U/ml XO by lucigenin chemiluminescence. Fig 2 Superoxide generation measured by lucigenin chemiluminescence in live human smooth muscle cells derived from normal or PE pregnancies. Determinations were performed in Krebs buffer containing NADPH (100M) and lucigenin (5M) over 3000s using the Chameleon II plate reader. PE cells showed a markedly greater level of superoxide generation than normal cells. Fig 3 To show that the Chameleon II was able to detect changes in superoxide generation in the absence of exogenous NADPH, normal smooth muscle cells were treated with ionomycin in Krebs buffer containing 5 mM lucigenin only. A transient but significant increase in O2- generation was detected by the high sensitivity reader.

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Superoxide (Lucigenin MLU)

LC+ is the lucigenin cation radical, and LCO2 is lucigenin dioxetane. In accord with this mechanism, O2- reduces lucigenin to its cation radical, which reacts with a second O2- to form the energy-rich dioxetane molecule emitting a photon (h) which is detected by the high sensitivity luminescence function of the Hidex Chameleon II plate reader. Cells can be stimulated to generate superoxide following an elevation of intracellular calcium levels which activate both NADPH oxidase activity and mitochondrial respiration.

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METHODS
Human umbilical artery smooth muscle cells from normal or preeclamptic (PE) pregnancies were cultured in Dulbeccos modified Eagles medium (DMEM) containing 20% serum in white 96 well microplates. When confluent, medium was changed to DMEM containing 1% serum for 24 h. Cells were then treated with warmed Krebs buffer containing 5 mM HEPES, 5M lucigenin, 100M NADPH and 2 M ionomycin, a calcium ionophore which induces cellular generation of O2. Chemiluminescence was detected in the Hidex Chameleon II plate reader (supplied by Cronus Technologies Ltd, UK) using the luminescence kinetics mode.

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DISCUSSION

Results from our evaluation of the Hidex Chameleon II plate reader revealed that the higher sensitivity of this model was essential for the measurement of small changes in superoxide generation from living vascular cells under culture conditions. Other fluorescent dyes sensitive to reactive oxygen species can be simultaneously detected by this plate reader to confirm findings with lucigenin chemiluminescence. The high sensitivity multi-technology capability of the Chameleon II makes it an ideal bench research apparatus for studies of oxidative stress in models of vascular, neurodegenerative and other diseases.