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Assay Method Information

Assay Name:  Inhibition Assay
Description:  Human HEK293 cells over-expressing human IK are grown in culture medium (DMEM supplemented with 10% foetal bovine serum), in polystyrene culture flasks (175 mm2) in a humidified atmosphere of 5% CO2 in air, at 37 C. Cell confluence should be 80-90% on day of plating. Cells are rinsed with 4 mL of PBS (phosphate buffered saline) and incubated 2 min with 1 mL of Trypsin-EDTA. After addition of 25 mL of culture medium cells are re-suspended by trituration with a 25 mL pipette.The cells are seeded at a density of 3106 cells/mL (25 μL/well) in black-walled, clear bottom, 384-well plates pre-treated with 0.01 g/L poly-D-lysin (20 μL/well for ≧30 min). Plated cells were allowed to proliferate for 24 h before loading with dye.BTC-AM (50 mg, Invitrogen) is added 25.5 μl DMSO. The BTC-AM stock solution (2 mM) is diluted to a final concentration of 2 μM in Cl+ free assay buffer (in mM: 140 Na+-gluconate, 2.5 K+-gluconate, 6 Ca2+-gluconate, 1 Mg2+ gluconate, 5 glucose, 10 HEPES, pH 7.3) containing 2 μM ouabain, 2 mM amaranth and 1 mM tartrazine.The culture medium is aspirated from the wells, and 25 μl of the BTC-AM loading solution are added to each well. The cells are incubated at 37 C. for 60 min.After the loading period, the Tl+-sensitive BTC fluorescence signal is measured over time using a FLIPR.
Affinity data for this assay
 

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Last update November 1, 2007
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