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A multi-level analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins

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Cortese-Krott, M., Rodriguez-Mateos, A., Kuhnle, G., Brown, G., Feelisch, M. and Kelm, M. (2012) A multi-level analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins. Free Radical Biology and Medicine, 53 (11). pp. 2146-2158. ISSN 0891-5849

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To link to this article DOI: 10.1016/j.freeradbiomed.2012.09.008

Abstract/Summary

Diaminofluoresceins are widely used probes for detection and intracellular localization of NO formation in cultured/isolated cells and intact tissues. The fluorinated derivative, 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM), has gained increasing popularity in recent years due to its improved NO-sensitivity, pH-stability, and resistance to photo-bleaching compared to the first-generation compound, DAF-2. Detection of NO production by either reagent relies on conversion of the parent compound into a fluorescent triazole, DAF-FM-T and DAF-2-T, respectively. While this reaction is specific for NO and/or reactive nitrosating species, it is also affected by the presence of oxidants/antioxidants. Moreover, the reaction with other molecules can lead to the formation of fluorescent products other than the expected triazole. Thus additional controls and structural confirmation of the reaction products are essential. Using human red blood cells as an exemplary cellular system we here describe robust protocols for the analysis of intracellular DAF-FM-T formation using an array of fluorescence-based methods (laser-scanning fluorescence microscopy, flow cytometry and fluorimetry) and analytical separation techniques (reversed-phase HPLC and LC-MS/MS). When used in combination, these assays afford unequivocal identification of the fluorescent signal as being derived from NO and are applicable to most other cellular systems without or with only minor modifications.

Item Type:Article
Refereed:Yes
Divisions:Faculty of Life Sciences > School of Chemistry, Food and Pharmacy > Department of Chemistry
Faculty of Life Sciences > School of Chemistry, Food and Pharmacy > Department of Food and Nutritional Sciences > Human Nutrition Research Group
ID Code:29434
Publisher:Elsevier

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