Microchip electrophoresis with laser-induced fluorescence detection for the determination of the ratio of nitric oxide to superoxide production in macrophages during inflammation
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It is well known that excessive production of reactive oxygen and nitrogen species is linked to the development of oxidative stress-driven disorders. In particular, nitric oxide (NO) and superoxide (O2 •−) play critical roles in many physiological and pathological processes. This article reports the use of 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate and MitoSOX Red in conjunction with microchip electrophoresis and laser-induced fluorescence detection for the simultaneous detection of NO and O2 •− in RAW 264.7 macrophage cell lysates following different stimulation procedures. Cell stimulations were performed in the presence and absence of cytosolic (diethyldithiocarbamate) and mitochondrial (2-methoxyestradiol) superoxide dismutase (SOD) inhibitors. The NO/O2 •− ratios in macrophage cell lysates under physiological and proinflammatory conditions were determined. The NO/O2 •− ratios were 0.60 ± 0.07 for unstimulated cells pretreated with SOD inhibitors, 1.08 ± 0.06 for unstimulated cells in the absence of SOD inhibitors, and 3.14 ± 0.13 for stimulated cells. The effect of carnosine (antioxidant) or Ca2+ (intracellular messenger) on the NO/O2 •− ratio was also investigated.
KeywordsBioanalytical methods Inflammation Macrophages Microchip electrophoresis Nitric oxide Superoxide
This study was funded by the National Science Foundation (CHE-1411993) and the National Institutes of Health (COBRE P20GM103638). G.C. gratefully acknowledges the support of an American Heart Association–Midwest Affiliate Postdoctoral Research Fellowship (NFP0075515). J.M.S. acknowledges the Madison and Lila Self Graduate Fellowship for support. We also thank Richard Piffer and Nancy Harmony for their helpful comments and editorial support.
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Conflict of interest
The authors declare that they have no conflict of interest.
Research involving human participants and/or animals
This article does not contain any studies with human participants performed by any of the authors.
- 1.Aaltoma SH, Lipponen PK, Kosma VM. Inducible nitric oxide synthase (iNOS) expression and its prognostic value in prostate cancer. Anticancer Res. 2001;21:3101–6.Google Scholar
- 10.Cooke JP. The pivotal role of nitric oxide for vascular health. Can J Cardiol. 2004;20(Suppl B):7B–15.Google Scholar
- 25.Culbertson CT. Single cell analysis on microfluidic devices. Methods Mol Biol. 2006;339:203–16.Google Scholar
- 40.Pou S, Pou WS, Bredt DS, Snyder SH, Rosen GM. Generation of superoxide by purified brain nitric oxide synthase. J Biol Chem. 1992;267:24173–6.Google Scholar
- 43.Caruso G, Fresta CG, Martinez-Becerra F, Antonio L, Johnson RT, de Campos RPS, Siegel JM, Wijesinghe MB, Lazzarino G, Lunte SM. Carnosine modulates nitric oxide in stimulated RAW 264.7 macrophages. Mol Cell Biochem. 2017. doi: 10.1007/s11010-017-2991-3.
- 45.Nicholls DG. Mitochondrial calcium function and dysfunction in the central nervous system. Biochim Biophys Acta. 1787;2009:1416–24.Google Scholar