| Assay Method Information | |
| | In Vitro Activity Test |
| Description: | Using the characteristics of luciferase binding to the substrate to generate a chemiluminescence reaction, the plasmid containing mineralocorticoid receptor (MR) ligand binding domain (LBD) fused with Gal4 DNA binding domain (DBD) and the firefly luciferase reporter gene plasmid under the control of Gal4 UAS (upstream activation sequence) are transfected into human embryonic kidney cells (HEK293). The changes in mineralocorticoid receptor activity before and after stimulation or the influence of different stimuli on mineralocorticoid receptor activity are judged by the level of firefly luciferase activity. At the same time, in order to reduce the impact of internal change factors on the accuracy of the experiment, the plasmid with Renilla luciferase gene is used as a control plasmid to transfect cells to provide an internal control for transcriptional activity, so that the test results are not interfered by changes in experimental conditions.Test Method1) The HEK293 cells were collected after trypsinization and the cell density was adjusted to 500,000 cells/mL;2) FuGENE HD transfection reagent was added to the cell suspension;3) The above cell suspension was inoculated into a 96-well cell culture plate of 100 μL/well, and incubated for 24 hours at 37° C., 5% CO2;4) A series of concentrations of the test compound solution and EC80 concentration of agonist aldosterone were added to each well and incubated for 18 hours;5) Firefly and Renilla luciferase signals were detected by Promega dual luciferase reporter gene test system.Result Processing:1) After obtaining the firefly luciferase signal (F) and the Renilla luciferase signal (R), the Renilla luciferase signal was used for correction, that is, the F/R value was used for subsequent calculation of inhibition rate;2)% inhibition rate=(Max−X)/(Max−Min)×100%, wherein Max is the F/R value of the positive control well, Min is the F/R value of the negative control well, and X is the test compound well of different concentrations F/R value;3) IC50 was calculated by GraphPrism 5.0 mapping software. |
| Affinity data for this assay | |
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