| Assay Method Information | |
| | In Vitro Assay |
| Description: | This potassium flux assay employs the cell-permeable, potassium-sensitive dye, IPG-2 AM, to quantify potassium ion flux through potassium channels.In general, TREX HEK 293 or HEK 293 cells were stably transfected with either an inducible or non-inducible expression vector containing the full-length cDNA coding for the desired human KV7.2/KV7.3 or in combination with another full-length cDNA for a second desired human KV7 potassium channel. Potassium channel-expressing cell lines were induced with tetracycline (1 g/mL), if required, and plated on 384-well poly-D-lysine (PDL)-coated plates in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation, culture media was removed and cells were loaded with 5 M IPG-2 AM dye for 1 hour in Assay buffer (140 mM NaCl, 20 mM RbCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer), 10 mM glucose, adjusted with Tris to pH 7.4). Excess dye was removed, and cells incubated at room temperature for 20 minutes with or without test compound. A Hamamatsu FDSS Cell was used to perform a 1:1 addition of K challenge buffer (150 mM NaCl, 10 mM HEPES, 2 mM CaCl2, 10 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4 for human KV7.2/KV7.3, and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength of 558 nm. Non-potassium channel-mediated potassium influx was determined in the presence of DMSO, and maximal influx was determined in the presence of a known KV7.x channel modulator. For each test compound, a concentration response curve was generated with 16 concentrations points, 2-fold serial dilution starting at 30 M and an EC50 value was determined. |
| Affinity data for this assay | |
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