Cell-based assay of IDO1 and TDO2 activities

HEK 293 cells were seeded in a six-well plate at a density of 5 × 10⁵ cells/well. The next day, the HEK 293 cells were transfected with pcDNA3.1-hIDO or pcDNA3.1-hTDO using Lipofectamine 2000 according to the manufacturer’s instructions. The cells were seeded in 96-well plates at a density of 2.5 × 10⁴ cells/well 24 h after transfection and treated with tested compounds. After an additional 12 h of incubation, 200 μL of the culture medium from each well was transferred to a 96-well plate and mixed with 100 μL of 30% trichloroacetic acid. The plate was incubated at 65 °C for 15 min to hydrolyze the N-formylkynurenine produced by the catalytic reaction of IDO1 or TDO2. The reaction mixture was then centrifuged at 12 000 r/min for 10 min. Then, 100 μL of the supernatant was transferred to another 96-well plate and mixed with 100 μL of 2% (w/v) p-dimethylaminobenzaldehyde in acetic acid. The yellow color derived from kynurenine was measured at 492 nm using a SpectraMax Plus 384 microplate reader (Molecular Devices, Sunnyvale, CA, USA).