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An experimental and theoretical study of DAF‐FM activation by NO: Toward calibration of an NO‐sensitive fluorescent dye
Author(s) -
Namin Shabnam M.,
Joshi Mahesh S.,
Bautista Carolina,
Nofallah Sara,
Tsoukias Nikolaos M.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.628.16
Subject(s) - fluorescence , umbilical vein , chemistry , biophysics , kinetics , calibration , analytical chemistry (journal) , in vitro , biochemistry , chromatography , biology , physics , optics , quantum mechanics
The quantitative measurement of NO in vitro has been hampered by the lack of a method that can detect nanomolar levels with spatial and temporal resolution and without interference from other reaction byproducts. 4,5 diaminofluorescein (DAF‐FM) is a sensitive NO fluorescence probe used widely for qualitative but not quantitative assessment of cellular NO production. With the aid of a mathematical model of NO/DAF‐FM reaction kinetics, we have conducted experimental studies to calibrate the fluorescence signal and estimate NO generation in different systems. Kinetic analysis suggests the following dependence of DAF‐FM triazole (DAF‐T) generation rate: DAF‐T fluorescence was measured in a series of experiments with varying DAF‐FM and NO donor concentrations (spermine/NO). Experiments showed that the slope of fluorescent intensity is proportional to [NO] 2 and exhibits a Monod type dependence on DAF‐FM concentration, thus validating the proposed kinetic mechanism. A calibration procedure was formed and applied in a cell culture system. Human umbilical vein endothelial cells (HUVEC) were stimulated with hemodynamic and agonist stimuli and the release rate of NO was assessed. [Supported by AHA grant NSDG043506N and NIH grant HL095101]