4.7. IDO enzyme assay

The IDO inhibitory activity assay was carried out as described in the reference with some modifications.38 In brief, 90 mL of the reaction mixture in a 96-well black plate contained 50 mM potassium phosphate buffer (pH 6.5), 20 mM ascorbic acid, 10 μM methylene blue, 100 μg mL^–1 catalase, 200 μM l-tryptophan and the test compounds (10 mM in DMSO diluted with 50 mM potassium phosphate buffer before use). The fluorescence (λ_ex 360 nm, λ_em 480 nm) was measured. The plate was incubated at 37 °C for 1 h after 10 μL of 20 μg mL^–1 IDO was added. To stop the reaction, 20 μL of 1 M NaOH solution was added and the plate was incubated at 60 °C for 15 min. The plate was placed at room temperature for about 1.5 h before the measurement of fluorescence (λ_ex 360 nm, λ_em 480 nm). The percentage of inhibition was reported as (100 – (A/B × 100)), where A is the IDO activity in the presence of the inhibitor and B is the IDO activity in the absence of the inhibitor. The IC₅₀ values were generated from 3 independent experiments with different concentration points. The results were calculated using GraphPad Prism 7.