| Assay Method Information | |
| | Measurement of Inhibitory Activity on AT1 Receptor (AT1R)/AT2 Receptor (AT2R) |
| Description: | Through the following steps, the inhibitory activity of the compound on AT1R/AT2R (IC50 value) was determined: 1) An appropriate amount of 1 TLB (Tag-lite Buffer) was prepared and well mixed for use. 2) The compound was diluted by 10 times with ddH2O or DMSO. The compound was then dilute to 4 times of the working concentration with IX TLB and mixed well for use. 3) 8600 nM Tag-lite angiotensin receptor red agonist was diluted to 12 nM (4 Kd) with 1 TLB. 4) 5 ml 1 TLB was taken into a 15 ml centrifuge tube. 5) After thawing 1 tube of Tb-labeled AT1R/AT2R cells in a 37 C. water bath, the cells were quickly transferred to the IX TLB in step 4), mixed gently, and centrifuged at 1200 g for 5 minutes at room temperature. 6) The supernatant was aspirated gently, and the cells were resuspended and mixed in 2.7 ml 1 TLB, and then placed at room temperature until use. 7) 10 μl cells were added to all test wells, and 5 μl 4 working solution of the compound from step 2) was added to the corresponding test wells. 5 μl 4 Tag-lite angiotensin receptor red agonist well diluted in step 3) was added to all test wells. 8) After leaving the reaction plate at room temperature for 1 h, data were measured and analyzed using Envision HTRF Reader, and the half inhibitory concentration (IC50) of the compound on AT1R/AT2R was calculated with the GraphPad Prism four-parameter equation. |
| Affinity data for this assay | |
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