| Assay Method Information | |
| | In Vitro Urat1 Assay |
| Description: | A plasmid (EX-T4563-M03, GeneCopoeia) containing the full-length human URAT1 gene (SLC22A12) was transfected into Flp-InT-REx-293 cells to construct URAT1 high-expression cell 293/hURAT1. The transfected cells were assayed for ability to uptake uric acid labelled with radioisotope. The test compounds were then evaluated for their activity by determining of their ability to block uric acid uptake by the transfected cells.293/h URAT1 cells were plated at a density of 40000 cells/well in poly D-lysine-coated 96-well plates (BD, 356461) and incubated overnight. The medium was removed and a pre-warmed reaction buffer was added (125 mM sodium gluconate, 4.8 mM potassium gluconate, 1.3 mM calcium gluconate, 1.2 mM potassium dihydrogen phosphate, 1.2 mM magnesium sulfate, 5.6 mM glucose, 25 mM HEPES, pH 7.4) before incubating at 37° C. for 10 minutes. The buffer was removed and another reaction buffer containing 50 μM 14C-uric acid (American Radiolabeled Chemicals, ARC0513) and the test compounds or solvent control was added before incubating at 37° C. for 5 minutes. The buffer was removed, and the plates were washed 3 times with the buffer. Cells were lysed by adding 100 mM NaOH for 20 minutes. Cell lysates were transferred to Isoplate-96 well plates (PerkinElmer, 6005040), mixed with liquid scintillators and were counted in a MicroBeta2 (PerkinElmer) counter.The test compounds were all dissolved in DMSO, and DMSO of the same concentration without the test compounds was used as a solvent control. The amount of uric acid uptake by the cells in the DMSO solvent control was taken as 100%, and the inhibition of uric acid uptake by cells in the test wells for each compound was calculated as a percentage. The IC50 of each compound was calculated using the percentage of inhibition at different concentrations. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |