Fluorescent Indicators for Imaging Nitric Oxide Production

Kojima, Hirotatsu; Urano, Yasuteru; Kikuchi, Kazuya; Higuchi, Tsunehiko; Hirata, Yasunobu; Nagano, Tetsuo

doi:10.1002/(sici)1521-3773(19991102)38:21<3209::aid-anie3209>3.0.co;2-6

Cited by 532 publications

(423 citation statements)

References 5 publications

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“…Nevertheless, and in addition to performing standard controls most investigators in this field will be familiar with, the use of analytical separation techniques capable of positively identifying the triazole product permits one to apply such probes with a fair degree of confidence for the detection of NO formation in cellular systems. 13 CͲ 1 H couplings from ɷ C 80.9 ppm to both hydrogens in the two (symmetrical) fluorinated aromatic rings (ɷ H 6.83 ppm and ɷ H 6.48 ppm) as well as to both hydrogens in the aromatic system which bears the ɶͲlactone moiety (ɷ H 6.65 ppm and ɷ H 6.23 ppm). C) HMBC spectrum with 13 C NMR spectrum superimposed on left hand side and 1 H NMR spectrum on top.…”

Section: Resultsmentioning

confidence: 99%

“…4ͲAminoͲ5ͲmethylaminoͲ2',7'Ͳdifluorofluorescein (DAFͲFM) is a fluorinated DAFͲ2 derivative with improved NOͲsensitivity, pHͲstability, and resistance to photoͲbleaching [13].…”

Section: Principlesmentioning

confidence: 99%

“…(1) Dissolve 1 mg DAFͲFM authentic standard in 0.5 ml d 6 ͲDMSO (2) Run 1 H NMR and 13 C NMR spectra of DAFͲFM The structure and complete NMR assignments of DAFͲFM (Figure 8 Panel A) were established on the basis of various NMR spectra ( 1 H, 13 C, HSQC, HMBC, 1 HͲ 1 H COSY and NOESY), which were acquired from a solution (1 mg/0.5 ml) in d 6 ͲDMSO at 700 MHz. The molecular formula for DAFͲFM is C 21 H 14 F 2 N 2 O 5 .…”

Section: Structural Analysis Of Dafͳfm By Nmrmentioning

confidence: 99%

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A multilevel analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins

Cortese‐Krott

Rodriguez-Mateos

Kuhnle

et al. 2012

Free Radical Biology and Medicine

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Diaminofluoresceins are widely used probes for detection and intracellular localization of NO formation in cultured/isolated cells and intact tissues. The fluorinated derivative, 4ͲaminoͲ5Ͳ methylaminoͲ2',7'Ͳdifluorofluorescein (DAFͲFM), has gained increasing popularity in recent years due to its improved NOͲsensitivity, pHͲstability, and resistance to photoͲbleaching compared to the firstͲ generation compound, DAFͲ2. Detection of NO production by either reagent relies on conversion of the parent compound into a fluorescent triazole, DAFͲFMͲT and DAFͲ2ͲT, 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 DAFͲFMͲT formation using an array of fluorescenceͲbased methods (laserͲscanning fluorescence microscopy, flow cytometry and fluorimetry) and analytical separation techniques (reversedͲphase HPLC and LCͲ 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.

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Section: Resultsmentioning

confidence: 99%

“…4ͲAminoͲ5ͲmethylaminoͲ2',7'Ͳdifluorofluorescein (DAFͲFM) is a fluorinated DAFͲ2 derivative with improved NOͲsensitivity, pHͲstability, and resistance to photoͲbleaching [13].…”

Section: Principlesmentioning

confidence: 99%

Section: Structural Analysis Of Dafͳfm By Nmrmentioning

confidence: 99%

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A multilevel analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins

Cortese‐Krott

Rodriguez-Mateos

Kuhnle

et al. 2012

Free Radical Biology and Medicine

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“…hIFN-g, hTNF-a and hIL-1b were purchased from Boehringer Mannheim (Tokyo, Japan). DAF-FM was synthesized according to the reported method in our own laboratories (Kojima et al, 1999). [ 14 C]-L-Arginine was purchased from NEN Life Science Products (Boston, MA, U.S.A.).…”

Section: Methodsmentioning

confidence: 99%

“…Prior to assay, the cells were seeded in 96-well plates (2610 5 cells per well) and allowed to attach overnight. NO production induced by exposure of the cells to 200 u ml 71 human interferon-g (hIFN-g), 10 ng human tumour necrosis factor-a (hTNF-a) and 2 ng human interleukin-1b (hIL-1b) for 24 h at 378C (Sherman et al, 1993) was measured with the recently described¯uorescent indicator DAF-FM (Kojima et al, 1999;Nakatsubo et al, 1998). A dimethyl sulphoxide (DMSO; ®nal concentration, 0.02%) solution of DAF-FM (1 mM) and the test compound at a concentration of 0.3 mM ± 1 mM were added at the same time as cytokines.…”

Section: Measurement Of No Production By Dld-1mentioning

confidence: 99%

Selective inhibition of human inducible nitric oxide synthase by S‐alkyl‐L‐isothiocitrulline‐containing dipeptides

Park

Higuchi

Kikuchi

et al. 2001

British J Pharmacology

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1 The aim of this study was to investigate the structure-activity relationship of S-alkyl-Lisothiocitrulline-containing dipeptides towards three partially puri®ed recombinant human nitric oxide synthase (NOS) isozymes, as well as the e ects of these compounds on cytokine-induced NO production by human DLD-1 cells. 2 In an in vitro assay, S-methyl-L-isothiocitrulline (L-MIT) was slightly selective for human neuronal NOS (nNOS) over the inducible (iNOS) or endothelial (eNOS) isozyme, but the combination of a hydrophobic L-amino acid (L-Phe, L-Leu or L-Trp) with L-MIT dramatically altered the inhibition pattern to give selective iNOS inhibitors. Introduction of a hydroxy, nitro, amino or methoxy group at the para position of the aromatic ring of L-MIT-L-Phe (MILF) decreased the selectivity and inhibitory potency. A longer or larger S-alkyl group also decreased the selectivity and potency. Dixon analysis showed that all of the dipeptides were competitive inhibitors of the three isoforms of human NOS. The enzymatic time course curves indicated that MILF was a slow binding inhibitor of human iNOS. 3 These results suggest that the human NOS isozymes have di erent-sized cavities in the binding site near the position to which the C-terminal of L-arginine binds, and the cavity of iNOS is hydrophobic. Interestingly, L-MIT-D-Phe (MIDF) showed little inhibitory activity or selectivity, suggesting that the cavity of human iNOS is located in a well-de®ned direction from the a carbon atom. 4 NO production in cytokine-stimulated human DLD-1 cells was measured with a¯uorescent indicator, DAF-FM. MILF, L-MIT-L-Trp(-CHO) (MILW) and L-MIT-L-Tyr (MILY) showed more potent activity than L-MIT in this whole-cell assay. 5 Thus, S-alkyl-L-isothiocitrulline-containing dipeptides are selective inhibitors of human iNOS, and work e ciently in cell-based assay.

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Novel Zinc Fluorescent Probes Excitable with Visible Light for Biological Applications

et al. 2000

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Fluorescent Indicators for Imaging Nitric Oxide Production

Cited by 532 publications

References 5 publications

A multilevel analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins

A multilevel analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins

Selective inhibition of human inducible nitric oxide synthase by S‐alkyl‐L‐isothiocitrulline‐containing dipeptides

Novel Zinc Fluorescent Probes Excitable with Visible Light for Biological Applications

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