| Assay Method Information | |
| | URAT1 In Vitro Inhibition Activity-Method B |
| Description: | HEK293-URAT1 cell Lines were donated by Japan Fuji Biomedical Research Institute. Negative control cell of HEK293 (MOCK cells) which was transfected with pcDNA3.1 empty vector. HEK293-URAT1 cell lines and MOCK cell lines were cultured in complete growth medium consisting of DMEM supplemented with 10% FBS, penicillin and streptomycin.Preparation of working solution: Each stock solutions was diluted to different concentrations (6, 20, 60, 200 and 600 μmol/L) with DMSO as 200× working solution, which was then diluted to 2×compound working solution with HBSS (Cl− free) buffer. Radiolabeled substrate 14C-Uric acid solution was diluted with HBSS (Cl− free) buffer to obtain 2× working solution which was mixed with an equal volume of 2× compound working solution to obtain the mixture of radiolabeled substrate and compound working solution.HER293-URAT1 and MOCK cells were seeded onto 24-well plates at the density of 1.5×106 cells per well. The cells were incubated at 37° C., 5% CO2 overnight. After cultured for approximately 2 to 3 days, cells were used for the experiments. The culture medium were removed from the wells, and cells were washed with HBSS (Cl− free) and incubated in 37° C. HBSS (Cl− free) for 10 min. HBSS was replaced with 500 μL of the mixture of radiolabeled substrate and compound working solution. The final concentration of 14C-Uric acid in the assay was 5.0 μmol/L. Plates were incubated at 37° C., 5% CO2 for 2 min, and the reaction was stopped by the addition of pre-chilled HBSS (Cl− free) by washing three times. 400 μL NaOH (0.1 mmol/L) was added to lyse the cells and the cell lysate was collected to scintillation vials, and 3 ml scintillant (Aquasol-2, PerkinElmer) was added and after mixing completely, the radioactivity was counted by Tri-Carb 2910TR liquid scintillation counter. Each concentration of compounds, positive control and negative control were repeated in two wells (n=2). Inhibition % data were calculated using the formula:Inhibition=[100×(U−U 0)/(U c −U 0)]%, and analyzed using Prism5 software.U0: Average of signals of MOCK cells;Uc: Average of signals of radiolabeled substrate. The half inhibition concentration of the tested compounds to URAT1 were analyzed using Prism 5 software. |
| Affinity data for this assay | |
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