ROS levels in neurons were detected by dichlorofluorescein diacetate assay (DCF-DA; Molecular Probes, Eugene, OR) as described [40]. Human primary neuronal cells cultured in 96-well plates (20,000 cells/well) for 14 days were loaded with DCF-DA (10 μM) for 40 min at 37 °C in 200 μl of cell culture media without phenol red in the presence or absence of specific inhibitors. After washing off the excess DCF-DA, neuronal cells were stimulated with test compounds with/without specific inhibitor for 30-120 min in an ELISA plate reader. The fluorescence intensity was then read at differential time points at excitation 488 nm and emission at 525 nm. A standard curve was generated with 6.25, 12.5, 25, 50, and 100 μM acetaldehyde. Results were expressed as mean relative fluorescence units per micromole of Ach 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 NOX/XOX-mediated ROS production was determined by the use of specific inhibitor to NOX (200 μM, apocynin) or XOX (200 μM, allopurinol). Expression of NOX/XOX protein was determined by Western blot analyses as described previously [14].
