title: A multilevel analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins.
abstract: 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 because of its improved NO sensitivity, pH stability, and resistance to photobleaching 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. Although 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.
authors.affiliation: Cardiovascular Research Laboratory, Department of Cardiology, Pneumology, and Angiology, Medical Faculty, Heinrich Heine University, Düsseldorf 40225, Germany. miriam.cortese@uni-duesseldorf.de
authors.forename: Miriam M
authors.initials: MM
authors.lastname: Cortese-Krott
authors.forename: Ana
authors.initials: A
authors.lastname: Rodriguez-Mateos
authors.forename: Gunter G C
authors.initials: GG
authors.lastname: Kuhnle
authors.forename: Geoff
authors.initials: G
authors.lastname: Brown
authors.forename: Martin
authors.initials: M
authors.lastname: Feelisch
authors.forename: Malte
authors.initials: M
authors.lastname: Kelm
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000592:standards; D056148:Chromatography, Reverse-Phase; D004912:Erythrocytes / Q000378:metabolism; D005434:Flow Cytometry; D005452:Fluoresceins / Q000737:chemistry* / Q000302:isolation & purification; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000302:isolation & purification; D005609:Free Radicals / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D018613:Microscopy, Confocal; D008968:Molecular Conformation; D009569:Nitric Oxide / Q000378:metabolism*; D010084:Oxidation-Reduction; D012015:Reference Standards; D059010:Single-Cell Analysis / Q000379:methods*; D013050:Spectrometry, Fluorescence; D053719:Tandem Mass Spectrometry
chemical_list: C503301:4-amino-5-methylamino-2',7'-difluorofluorescein diacetate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005609:Free Radicals; D009569:Nitric Oxide