| Assay Method Information | |
| | IDO2 Assay |
| Description: | The reference compound INCB024360 (a potent and selective indoleamine 2,3-dioxygenase (IDO1) inhibitor) and test compounds were diluted from source stock solutions, and 20 μL of diluted compound was added to each plate, which contained 180 uL culture medium. Serial dilution was performed to obtain different concentrations of compound according to the plate the plate map, and the assay plate was incubated at 37° C./5% CO2 for 72 h. After incubation, plates and reagents were allows to equilibrate to room temperature. From the assay plate, 140 μL of the supernatant was transferred to a new plate using multichannel pipette. Trichloroacetic acid powder was dissolved in ddH2O to prepare 6.1N trichloroacetic acid. 10 μL of 6.1N trichloroacetic acid was added into each well, mix the content using a plate shaker at 300-500 rpm for 30 seconds. The plates were incubated at 55° C. for 40 minutes, and then centrifuged for 10 min at 2500 rpm. Following centrifugation, 100 μL of the supernatant was transferred to another 96 plate, and 100 μL of 2% (w/v) 4-dimethylaminobenzaldehyde in acetic acid was added into each well. Measurement of the signal was performed using Envision at 480 nm. Throughout the foregoing process, care was taken to prevent exposure to light. |
| Affinity data for this assay | |
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