| Assay Method Information | |
| | HEK-URAT1 assay |
| Description: | URAT1 stably expressed HEK293 cells (HEK-URAT1) or HEK293 cells transfected with empty vector (HEK-mock) were cultured in DMEM culture medium supplemented with 10% fetal bovine serum in an incubator under conditions of 5% carbon dioxide at 37° C. Subsequently, 1×105 cells were respectively seeded in 24-well culture plate coated with poly D-lysine and the uptake test was started after the culturing for 3 days.The uptake experiment of uric acid was performed at 37° C. The cells were washed 3 times with a solution pH 7.4 (Hanks buffer solution that does not contain chloride ions, 125 mM sodium gluconate, 4.8 mM potassium gluconate, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.3 mM calcium gluconate, 25 mM HEPES, 5.6 mM glucose, and 12.4 mM tris) which was warmed to 37° C. for uptake experiment and equilibrated at 37° C. for 10 minutes. After a buffer solution was removed from the cells, 0.5 mL of an uptake solution containing 5 μM [14C] uric acid with or without a test compound was respectively added and the cells were incubated for 2 minutes. The cell uptake was stopped by adding an ice-cold Hanks buffer solution without chloride ions and the cells were washed 3 times. The cells were lysed with 0.1 N sodium hydroxide and the radioactivity was measured using LSC6100 (Aloka Co., Ltd. Tokyo). Each uptake into HEK-URAT1 was acquired by subtracting uptake into HEK-mock cells from the value of HEK-URAT1, converting the result to per mg protein of cells, and setting the value from uptake without the test compound as 100%. The uptake in each condition was performed in triplicate and each value was represented by averageħstandard deviation.(2) Test Results |
| Affinity data for this assay | |
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