| Assay Method Information | |
| | Evaluation of GLUT9 Inhibitory Activity |
| Description: | Uricase/Hyper transiently-transfected human GLUT9 stably expressing cells or mock cells (blank) were seeded in a 96 well plate (Corning) at 1.6×105 cells/well, and cultured overnight at 37° C., 5% CO2. D-MEM/high glucose (Wako Pure Chemical Industries, Ltd.) containing 10% Fetal Bovine Serum (Lifetechnology) and 100 units/ml penicillin/100 μg/ml streptomycin (GIBCO) was used as a medium. High K− buffer (129.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4.7H2O, 1.3 mM CaCl2. 2H2O, 25 mM HEPES, pH 7.4 with 1 M Tris) and the medium were mixed in equal amount to prepare Assay Buffer. The medium in each well was removed, and the test compound solution (final 1% DMSO) diluted with Assay Buffer was added thereto at 50 μl/well, and the mixture was left stand at room temperature for 30 to 60 min. For the solvent control and blank, Assay Buffer containing DMSO alone was added at 50 μl/well, and the mixture was left stand at room temperature for 30 to 60 min. In addition, uric acid solution (containing [14C]uric acid as a tracer) diluted with Assay Buffer was added to each well at 15 μl/well (final 300 μM uric acid), and the uptake reaction was performed at room temperature for 6 min. After the completion of the reaction, the cells were washed three times with ice-cooled Wash Buffer (Hank's Balanced Salt Solution containing 0.01% Bovine Serum Albumin) at 150 μl/well, and 0.1N aqueous NaOH solution was added thereto at 25 μl/well to dissolve the cell. MicroScint-20 (Perkin-Elmer) was added thereto at 150 μl/well, the plate was shaked, and CPM of [14C] was measured by TopCount NXT (Perkin-Elmer).Data was obtained by deducting average of CPM in blank well from average of CPM in each treated well. The inhibitory rate of the test compound in each concentration was calculated from the following formula: [(A−B)/A]×100, A is data of solvent control, B is data of test compound treatment. IC50 value (50% inhibition concentration) of the test compound was obtained by applying the inhibitory rate of the test compound in each concentration to logistic curve. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |