| Assay Method Information | |
| | Inhibitory Activity on A2A and A2B Receptors Assay |
| Description: | The screening procedure was as follows:1. adjusting the cell density to 6×105 cells/mL with serum-free medium;2. adding 5 mL of cytosol, 2.5 mL of NECA (Sigma, 119140-10MG), and 2.5 mL of compound solution sequentially to each well of a 384-well plate (Greiner Bio-One, 784075), in which the final concentration of NECA is 1 μM (CHO-K1/ADORA2A) or 0.1 μM (CHO-K1/ADORA2B), and the final concentration of the compound starts from 3 μM to triple dilutions less than 3 μM;3. placing the 384-well plate in 37° C. incubator for 30 min;4. adding 5 μL of cAMP-d2 and 5 μL of cAMP-ab (Cisbio, 62AM4PEB) sequentially;5. leaving the 384-well plate at room temperature for 1 h, protected from light; and6. reading the plate (Victor X5, PerkinElmer) and analyzing the data by XLfit nonlinear regression to calculate the IC50 of the compound. |
| Affinity data for this assay | |
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