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

A multi-level analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins
A multi-level analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins
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.
nitric oxide, fluorescence imaging, daf-fm, daf-2, nitrosation, red blood cells
0891-5849
2146-2158
Cortese-Krott, Miriam M.
7dc9b44c-847c-4196-8866-a3cc0c1dc357
Rodriguez-Mateos, Ana
874f6e8a-7b38-44af-b32b-4fdbb3019208
Kuhnle, Gunter
86ab1ce4-315f-4408-9178-62f0d20149e7
Brown, Geoff
60f8ce2c-b574-4079-953c-398f8783956b
Feelisch, Martin
8c1b9965-8614-4e85-b2c6-458a2e17eafd
Kelm, Malte
db2bb062-32d7-4b50-9f65-8ba89ffa5f42
Cortese-Krott, Miriam M.
7dc9b44c-847c-4196-8866-a3cc0c1dc357
Rodriguez-Mateos, Ana
874f6e8a-7b38-44af-b32b-4fdbb3019208
Kuhnle, Gunter
86ab1ce4-315f-4408-9178-62f0d20149e7
Brown, Geoff
60f8ce2c-b574-4079-953c-398f8783956b
Feelisch, Martin
8c1b9965-8614-4e85-b2c6-458a2e17eafd
Kelm, Malte
db2bb062-32d7-4b50-9f65-8ba89ffa5f42

Cortese-Krott, Miriam M., Rodriguez-Mateos, Ana, Kuhnle, Gunter, Brown, Geoff, Feelisch, Martin and Kelm, Malte (2013) A multi-level analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins. Free Radical Biology and Medicine, 53, 2146-2158. (doi:10.1016/j.freeradbiomed.2012.09.008). (PMID:23026413)

Record type: Article

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 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.

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2012 FRBM Methods paper-1_Oct_2012 Accepted MS.pdf - Other
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e-pub ahead of print date: 28 September 2012
Published date: 2013
Keywords: nitric oxide, fluorescence imaging, daf-fm, daf-2, nitrosation, red blood cells
Organisations: Faculty of Medicine

Identifiers

Local EPrints ID: 343571
URI: http://eprints.soton.ac.uk/id/eprint/343571
ISSN: 0891-5849
PURE UUID: a0435c97-8279-4df4-9c0b-ca7b7cd7f732
ORCID for Martin Feelisch: ORCID iD orcid.org/0000-0003-2320-1158

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Date deposited: 04 Oct 2012 15:56
Last modified: 18 Feb 2021 17:18

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Contributors

Author: Miriam M. Cortese-Krott
Author: Ana Rodriguez-Mateos
Author: Gunter Kuhnle
Author: Geoff Brown
Author: Martin Feelisch ORCID iD
Author: Malte Kelm

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