| Assay Method Information | |
| | Inhibitory Activity Assay |
| Description: | human ROMK and rat ROMK: 1. Materials and Instruments (1) FluxOR potassium ion channel assay (F10016, Invitrogen) (2) Ouabain (03125-1G, Sigma) (3) FlexStation3 microplate reader (Molecular Devices) (4) Human ROMK/HEK293 cell: HEK293 cell line stably expressing the ROMK channel transfected by human ROMK cDNA (NCBI SEQ ID NO. NM-000220.4) (5) Rat ROMK/HEK293 cell: HEK293 cell line transfected by rat ROMK cDNA (NCBI SEQ ID NO. NM-017023.1) stably expressing the ROMK channel (6) HEK293 cell line: Cell Bank of Chinese Academy of Sciences, GNHu43 2. Experimental Procedure - Human ROMK/HEK293 cell was seeded on PDL(Poly-D-lysine) coated plates at 20000 cells/well on the previous day; After overnight culture, the plate medium was discarded; then according to the Fluxor Potassium Ion Channel Assay Kit instructions, the dye was added at 100 μL/hole, and then incubated for 90 mins at room temperature; The dye was then decanted and 100 μL of assay buffer containing ouabain (300 μM) and probenecid were added in each well;1 μL of compound or DMSO was added to the corresponding wells, shocked for 30 seconds, and incubated for 30 mins at room temperature; The plates were placed in a FlexStation3 microplate reader, and then added with stimulation buffer (K2SO4:Tl2SO4:1XFluxOR Chloride-free Buffer:ddH2O=3:12:40:125) at 25 μL/well, then the value was read continuously for 5 mins at EX/EM of 490/525 nm immediately; and The IC50 of the present compounds on human ROMK channel was obtained by data processing software Graphpad. |
| Affinity data for this assay | |
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