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

[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.

[学科分类] 生物化学/生物物理

[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