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Development of a fluorescent indicator for nitric oxide based on the fluorescein chromophore.

Chemical & pharmaceutical bulletin (1998-03-21)
H Kojima, K Sakurai, K Kikuchi, S Kawahara, Y Kirino, H Nagoshi, Y Hirata, T Nagano
ABSTRACT

Endogenous nitric oxide (NO) appears to modulate many physiological and pathophysiological processes. In order to obtain direct evidence for NO functions in vivo, we have developed 4,5-diaminofluorescein (DAF-2) as a novel fluorescent indicator for NO. Green-fluorescent triazolofluorescein formed by the reaction of NO and DAF-2 affords high sensitivity for NO (detection limit: 5 nM). Membrane-permeable DAF-2 diacetate (DAF-2 DA) was loaded into activated rat aortic smooth muscle cells, where the ester bonds are hydrolyzed by intracellular esterase, generating DAF-2. The fluorescence in the cells increased in a NO concentration-dependent manner. This imaging method should be useful for studies of the dynamic biological actions of NO at the molecular level with fine temporal and spatial resolution.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
4,5-Diaminofluorescein diacetate solution, 5 mM in DMSO, ≥97% (HPLC)
Sigma-Aldrich
4,5-Diaminofluorescein diacetate, ≥98% (HPLC)
Sigma-Aldrich
5,6-Diaminofluorescein diacetate, BioReagent, suitable for fluorescence, 98% (HPCE)
Sigma-Aldrich
4,5-Diaminofluorescein, 5 mM in DMSO, ≥97% (HPLC)
Sigma-Aldrich
Nitric Oxide Synthase Detection System, Fluorimetric, sufficient for 200 reactions

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