| Assay Method Information | |
| | thallium-flux assay |
| Description: | The thallium flux assay is an in vitro method of measuring conductance through a potassium ion channel. Potassium channels are also permeable to thallium ions. The modulation of a K+ channel will thus increase or decrease thallium ion flow through the channel and thus, alter the observed fluorescence of a thallium-specific indicator dye.Stably transfected T-Rex-HEK-293-AnKir1 cells were cultured overnight in 384-well plates in media containing DMEM, 10% dialyzed FBS, and 1 μg/mL tetracycline to induce channel expression. The next day the cell culture medium was replaced with a dye-loading solution containing assay buffer (Hanks Balanced Salt Solution with 20 mM HEPES, pH 7.3), 0.01% (w/v) Pluronic F-127 (Life Technologies, Carlsbad, Calif.), and 1.2 μM of the thallium-sensitive dye Thallos-AM (TEFlabs, Austin, Tex.). After 1 hr incubation at room temperature, the dye-loading solution was washed from the plates and replaced with 20 μL/well of assay buffer. The plates were transferred to a Hamamatsu Functional Drug Screening System 6000 (FDSS6000; Hamamatsu, Tokyo, Japan), where 20 μL/well of each of the test compounds in assay buffer was added and allowed to incubate with the cells for 20 min. After incubation, a baseline recording was collected at 1 Hz for 10 s (excitation 470±20 nm, emission 540±30 nm); a thallium stimulus buffer was then added (10 μL/well), and data were then collected for an additional 4 min. The Tl+ stimulus buffer contained (in mM): 125 NaHCO3, 1.8 CaSO4, 1 MgSO4, 5 glucose, 12 Tl2SO4, 10 HEPES, pH 7.4. For Tl+ flux assays on Kir2.x, Kir4.1 and Kir6.2/SUR1 expressing cells, the Tl+ stimulus buffer contained 1.8 mM Tl2SO4. To ensure the small-molecule vehicle DMSO had no direct effect on AnKir1-dependent Tl+ flux, the assay's tolerance to different doses of DMSO was evaluated. The robustness and reproducibility of the assay was determined by comparing Tl+ flux through tetracycline-induced and tetracycline-free cells. |
| Affinity data for this assay | |
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