| Solution Information | help | |
| Enzyme: | Solute carrier family 12 member 5 | |
| inhibitor: | BDBM44165 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | METHOD: 1. Human embryonic kidney cells expressing KCC2 were plated at 20,000 cells/well in Dulbecco's modified medium (DMEM), 41.6% F12 (Gibco catalog 11320-033) in 384 well plate, black, clear bottom, poly-D-lysine coated (Greiner catalog 781946). 2. Cells were incubated overnight at 37 degrees C in 5%CO2. 3. Cells were loaded with 0.5 micromolar FluoZin2-AM dye (Invitrogen catalog number F24189) in assay buffer (Hanks Buffered Salt Solution, 20 mM HEPES, 0.2 mM oubain, 2mM bumetanide) for 48 minutes. 4. Dye was removed and the plate imaged using the Hamamatsu FDSS kinetic plate reader equipped with 480 nanometer excitation and 540 nanometer emission filters. 5. Compounds were tested in triplicate as 11-point 3-fold dilutions starting at 30 uM. 6. Thallium buffer stimulus (125 mM sodium bicarbonate, 12 mM thallium sulfate, 1 mM magnesium sulfate, 1.8 mM calcium sulfate, 5 mM glucose, 10 mM HEPES, pH 7.3.) was added and images collected at 1 Hz. 7. Assay buffer containing DMSO (0 | |
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