DAF-2 is a fluorescent detector for nitric oxide (NO) in cells and tissues with excitation and emission wavelengths of 495 and 517nm, respectively. In oxygenated solutions, reactive nitrogen species derived from NO autoxidation nitrosate DAF-2 to yield the highly fluorescent DAF-2 triazole[1]. The ability of DAF-2 to detect NO was strongly affected by the divalent cation concentration in the medium especailly calcium, as well as the incident light[2]. Once DAF-2 is fully excited, the fluorescence intensity is very stable for more than 30min but the existance of catecholamines and reducer like ascorbate attenuate the NO-induced fluorescence of DAF-2 mainly through an anti-oxidative action[3].
In vitro, DAF-2 (5µM) reliably quantified 4-bromo-A23187-stimulated NO from human EA.hy 926 endothelial cells after incubated 45min at 37°C. The fluorescence increase was detected immediately by HPLC-FLD[4]. 1mM DAF-2 incubated coronal slices of midbrain or hippocampus isolated from young adult rats for 30min at 33°C. Illumination at 450–490nm revealed punctate fluorescence in neurones in the lateral tegmental nucleus, dorsal raphe nucleus, dorsolateral periaqueductal grey matter, deep collicular layers and cortical areas[5].
References:
[1] Jourd'heuil D. Increased nitric oxide-dependent nitrosylation of 4,5-diaminofluorescein by oxidants: implications for the measurement of intracellular nitric oxide. Free Radic Biol Med. 2002 Sep 1;33(5):676-84.
[2] Broillet M, Randin O, Chatton J. Photoactivation and calcium sensitivity of the fluorescent NO indicator 4,5-diaminofluorescein (DAF-2): implications for cellular NO imaging. FEBS Lett. 2001 Mar 2;491(3):227-32.
[3] Nagata N, Momose K, Ishida Y. Inhibitory effects of catecholamines and anti-oxidants on the fluorescence reaction of 4,5-diaminofluorescein, DAF-2, a novel indicator of nitric oxide.J Biochem. 1999 Apr;125(4):658-61.
[4] Uhlenhut K, Högger P. Pitfalls and limitations in using 4,5-diaminofluorescein for evaluating the influence of polyphenols on nitric oxide release from endothelial cells. Free Radic Biol Med. 2012 Jun;52(11-12):2266-75.
[5] Brown L A, Key B J, Lovick T A. Bio-imaging of nitric oxide-producing neurones in slices of rat brain using 4,5-diaminofluorescein. J Neurosci Methods. 1999 Oct 15;92(1-2):101-10.
DAF-2是一种用于细胞和组织的NO荧光探针，激发/发射波长分别为495和517nm。在含氧溶液中，NO自氧化生成的活性氮物种使DAF-2发生亚硝化，生成高荧光的DAF-2triazole[1]。培养基中的二价阳离子（尤其是钙离子）以及入射光强烈影响DAF-2检测NO的能力[2]。DAF-2完全激发后，荧光强度在30min以上非常稳定，但儿茶酚胺和还原剂（如抗坏血酸）通过抗氧化作用显著减弱NO诱导的DAF-2荧光[3]。
在体外实验中，DAF-2（5μM）在37℃下孵育45分钟后能可靠地定量人EA.hy926内皮细胞受4-bromo-A23187刺激产生的NO，荧光增加立即通过HPLC-FLD检测到[4]。 1mM DAF-2于33°C下孵育年轻成年大鼠中脑或海马冠状切片30分钟，450-490nm波长下照明可见侧纹状体核、背外侧延髓、背外侧脑桥灰质、深层丘脑层和皮层区域神经元中的点状荧光[5]。