| Assay Method Information | |
| | Biological Assay |
| Description: | The Inhibitory Activity of the Present Compounds on Human ROMK and Rat ROMK ChannelsThe method described hereafter was used for determining the inhibitory activity of the present compounds on human ROMK and rat ROMK channels.1. Materials and Instruments(1) FluxOR potassium ion channel assay (F10016, Invitrogen)(2) Ouabain (O3125-1G, 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, GNHu432. Experimental ProcedureExcept for ddH2O and Ouabain, all of the experimental reagents are from FluxOR Potassium Ion Channel Assay Kit and the formulation methods also refer to the kit instructions. (1) Human ROMK/HEK293 cell was seeded on PDL (Poly-D-lysine) coated plates at 20000 cells/well on the previous day; (2) 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; (3) The dye was then decanted and 1004, of assay buffer containing ouabain (30004) and 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: 1×FluxOR 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 (6) 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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