| Assay Method Information | |
| | THR Reporter Gene Method |
| Description: | Huh7 cells were cultured in a DMEM medium supplemented with 10% FBS. The cells were inoculated into a 10 cm cell culture dish, proliferated to about 90% confluency, and co-transfected by using a liposome (Lipofectamine 2000) with human THRa eukaryotic expression plasmid or human THRβ eukaryotic expression plasmid, as well as reporter gene plasmid PGL4.26-DR4-Luc containing THR response sequence driver. The operation steps were carried out according to the instructions of Lipofectamine 2000. The next day after transfection, the cells were inoculated with a phenol red-free DMEM (supplemented with 5% activated carbon-treated FBS) into a 96-well cell culture plate at a seeding density of 20,000 cells per well and a volume of 135 μL per well. The cells adhered to wall 6 hours after the inoculation. Compounds dissolved in DMSO were diluted in phenol red-free DMEM (supplemented with 5% activated carbon-treated FBS) to be 20 to 10 times of the final concentration, and added to the cell wells at 15 μL per well, that is, the compounds were diluted 10 times again to reach the final concentration. Triiodothyronine T3 (100 nM) was set as a positive control, and 0.5% DMSO was set as a blank control. After the compounds were added, the cells were cultured at 37 C. in a 5% CO2 incubator overnight (16 hours). After incubation, the culture medium was discarded, and each well was added with 35 μL of serum-free and phenol red-free DMEM and 35 μL of Steady-Glo, shaked for 10 minutes in the dark, and the chemiluminescence value of the sample was detected. The agonistic activity of the compound was calculated as follows: % effect=(compound−blank control)/(positive control−blank control) 100%. The EC50 of the compound was obtained by fitting the agonistic activity of the compound and the logarithmic value of the compound concentration with GraphPad Prism. The lower the EC50 value, the better the activity. |
| Affinity data for this assay | |
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