NO levels in human neuronal culture were detected by the diaminofluorescein-2 diacetate assay (Molecular Probes) as described [41]. Removal of the acetate group from DAF-2DA by intracellular esterases produces a highly fluorescent DAF detected at excitation 488 nm and emission at 515 nm. Briefly, human neurons cultured in 96-well plates (20,000 cells/well) for 14 days were loaded with DAF-2DA (10 μM) for 40 min at 37 °C in 200 μl of cell culture media without phenol red with or without specific inhibitor. After removing the excess DCF-DA, neurons were stimulated with test compounds with/without specific inhibitor for 30-120 min, followed by differential time point fluorescence readings at excitation 488 nm and emission at 515 nm. A standard curve was generated with 1, 5, 10, 20, 50, and 100 μM SNAP (S-nitroso-N-acetylpenicillamine). Results were expressed as mean relative fluorescence units per micromole SNAP after subtracting blank values (media alone) from samples and standard values. The effect of EtOH/Ach (17.5 mM EtOH or 10 μM Ach) on iNOS induction in neurons was determined by NO production using the iNOS-specific inhibitor, L-NAME (50
