Reliable in vitro measurement of nitric oxide released from endothelial cells using low concentrations of the fluorescent probe 4,5‐diaminofluorescein

FEBS Letters

Müller, Christian¹  Räthel, Thomas R¹  Dirsch, Verena M¹  Vollmar, Angelika M¹  Leikert, Jürgen F¹ 

[1] Department of Pharmacy, Center of Drug Research, University of Munich, Butenandtstr. 5–13, D-81377 Munich, Germany

关键词: Nitric oxide;    4;    5-Diaminofluorescein;    Fluorescence spectroscopy;    Endothelial nitric oxide synthase;    DAF-2;    4;    5-diaminofluorescein;    L-NMMA;    N G-monomethyl-L-arginine;    NO;    nitric oxide;    NOC-9;    MAHMA·NONOate;    PBS;    phosphate-buffered saline;    PMA;    phorbol-12-myristate-13-acetate;   

DOI  :  10.1016/S0014-5793(01)02901-5

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

4,5-Diaminofluorescein (DAF-2) and its membrane-permeable derivate DAF-2 diacetate are fluorescent probes that have been developed to perform real-time biological detection of nitric oxide (NO). Their use for intracellular imaging, however, has recently been seriously questioned and data using DAF-2 for extracellular NO detection at low levels, as for example released from endothelial cells, are rare. Here we show that a reliable detection of low levels of NO in biological systems by DAF-2 is possible (a) by using low DAF-2 concentrations (0.1 μM) and (b) by subtracting the DAF-2 auto-fluorescence from the measured total fluorescence. The described method allows easy real-time detection of endothelial NO formation.

【 授权许可】

Unknown