Nitric oxide (NO) research in biomedicine has been hampered by the absence of a method that will allow quantitative measurement of NO in biological tissues with high sensitivity and selectivity, and with adequate spatial and temporal resolution. 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) is a NO sensitive fluorescence probe that has been used widely for qualitative assessment of cellular NO production. However, calibration of the fluorescent signal and quantification of NO concentration in cells and tissues using fluorescent probes, have provided significant challenge. In this study we utilize a combination of mathematical modeling and experimentation to elucidate the kinetics of NO/DAF-FM reaction in solution. Modeling and experiments suggest that the slope of fluorescent intensity (FI) can be related to NO concentration according to the equation: ddt[FI]=2αk1false[NOfalse]2[O2][DAF-FM]k[NO]+[DAF-FM] where α is a proportionality coefficient that relates FI to unit concentration of activated DAF-FM, k1 is the NO oxidation rate constant, and k was estimated to be 4.3 ± 0.6. The FI slope exhibits saturation kinetics with DAF-FM concentration. Interestingly, the effective half-maximum constant (EC50) increases proportionally to NO concentration. This result is not in agreement with the proposition that N2O3 is the NO oxidation byproduct that activates DAF-FM. Kinetic analysis suggests that the reactive intermediate should exhibit NO-dependent consumption and thus NO2• is a more likely candidate. The derived rate law can be used for the calibration of DAF-FM fluorescence and the quantification of NO concentration in biological tissues.

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]