| Assay Method Information | |
| | In Vitro Assay |
| Description: | In vitro URAT1 assay can be used to identify compounds having potential activity for decreasing serum uric acid. In a suitable test, the vectors that encode human URAT1 (URAT1 cDNA: Guangzhou Copoeia EX-T4563-M02) were used to transfect cells (human embryonic kidney cells, HEK293: Cell Bank of the Chinese Academy of Sciences, GNHu18). The transfected cells HEK293/hURAT1 cells were obtained, then their uptake ability of radiolabeled uric acid was determined. The activity of the compounds as URAT1 inhibitors can be evaluated by the ability of the compounds to block the uptake of uric acid in the transfected cells.The HEK293/hURAT1 cells in Eagle's minimal essential medium (EMEM) were inoculated in a 48-well plate that was coated with poly-D-lysine (Becton Dickinson, Catalog No. 356509), with an inoculation density of 105 cells/well, and incubated overnight. A reaction solution containing 14C uric acid (American Radioactive Compound, Catalog No. ARC 0513A) with a final concentration of 11.57 μM was prepared by the use or non-use of the test compounds in Hanks balanced salt solution (HBSS). The Hanks balanced salt solution (HBSS) contained 125 mM sodium gluconate, 4.8 mM potassium gluconate, 1.2 mM potassium dihydrogen phosphate, 1.2 mM magnesium sulfate, 1.3 mM calcium gluconate, 5.6 mM glucose and 25 mM HEPES (pH 7.3). After the medium was washed with the wash buffer (125 mM sodium gluconate, 10 mM HEPES, pH 7.3) for one time, the reaction solution prepared from the above step was added to each well and incubated at room temperature for 12 minutes. Then the reaction solution was removed, the cells were washed twice with the wash buffer and lysed with 0.2 M NaOH for 5 minutes. The cell lysate was transferred to a 96-well culture plate with a scintillation fluid (PerkinElmer, Catalog No. 1450-401), and counting of radioactivity was carried out on a Microbeta counter (PerkinElmer). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |