The chemiluminescent assay is used to confirm PCSEE MAO-A and MAO-B inhibitory effects and to test BNN and BVN hMAO-A and hMAO-B inhibition using MAO-Glo kit. Each enzyme\'s Arbitrary Light Unit (ALU) is measured in the presence of PCSEE, BNN, BVN, and standard DEP as an MAO-BI positive control. Briefly, hMAO-A and hMAO-B isozymes are diluted to 2× with reaction buffer (pH 7.4) and preincubated with 4× PCSEE, BNN, BVN, or DEP working solutions at RT for 30 min in white opaque 96-well plates. For determining activity inhibition, final 8.5 μg/mL concentrations of PCSEE, BNN, BVN, and DEP are used. For IC50 determination, 8× PCSEE and BNN working solutions are serially diluted using reaction buffers (pH 7.4) to make a 4× concentration. Ten points\' range of PCSEE (1.0 to 250.0 μg/mL) and BNN (up to 400 μM (135.4 μg/mL)) final concentrations is used. Controls used are with and without ethanol. Ethanol solvent in controls is kept to a maximum final (volume) of ≤2%. Each isozyme is substituted with the reaction buffer for the blank. Based on our preliminary optimizations and Valley\'s method, the reaction is initiated by adding 4× luciferin derivative substrate (LDS) for a final (concentration) of 40 and 4 μM for hMAO-A and hMAO-B reactions, respectively. The final volume per well of each reaction is 50 μL. The reaction is optimized for the amount of A and B enzyme used to be incubated for less than 3.5 h at RT. To stop the reaction and produce the luminescence signal RLDR is added to all wells, 50 μL to each well, and incubated for a further 30 min.
MCE has not independently confirmed the accuracy of these methods. They are for reference only.
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