| Assay Method Information | |
| | Inhibitory Activity Assay |
| Description: | hERG: 1. Materials and Instruments (1) FluxOR potassium ion channel assay (F10016, invitrogen) (2) FlexStation3 microplate reader (molecular devices) (3) hERG/HEK293 cell: HEK293 cell line stably expressing the hERG channel transfected by hERG cDNA (NCBI SEQ ID NO. NM-000238(RC215928, origene)). 2. Experimental Procedure (1) Human hERG/HEK293 cell was seeded on PDL(Poly-D-lysine) coated plates at 25000 cells/well on the previous day; (2) After overnight culture, the plate medium was discarded; then according to FluxOR potassium ion channel detection requirements operation, the dye was added at 100 μL/hole, and then incubated for 90 mins at room temperature; (3) The dye was then decanted and 100 μL of assay buffer containing 100 μL probenecid were added in each well; (4) 1 μL of compound or DMSO was added to the corresponding wells, shocked for 30 seconds, and incubated for 30 mins at room temperature; (5) The plates were placed in a FlexStation3 microplate reader, and then added with stimulation buffer (K2SO4:Tl2SO4:1XFluxOR Chloride-free Buffer:ddH2O=2:1:2:5) at 25 μL/well, then the value was read continuously for 5 mins at EX/EM of 490/525nm immediately; and (6) The IC50 of the present compounds on human hERG ion channel was obtained by data processing software Graphpad. |
| Affinity data for this assay | |
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