| Assay Method Information | |
| | Biological Assay |
| Description: | On the day of the assay, the reagents were prepared following the FLIPR® Potassium Assay Kit manual: prepare 2× dye solution, dilute the dye with assay buffer (20 mM HEPES in 1× HBSS, PH7.4), addition of probenecid to a final concentration of 5 mM, and vortexed vigorously for 1-2 minutes. The cell plate was flicked to remove medium and tapped on paper towels to remove excess media. The assay buffer and 2× dye solution were mixed 1:1 and added to each well for a total volume of 201 per well. The cell plate was moved to a plate shaker, agitated at 600 rpm for two minutes, and then incubated at 25° C. for one hour.The compounds were prepared in DMSO and transferred to a 384-well compound plate (PP, low binding), referred to as a source plate. Reference agonist (300 nM) compound and test inhibitor (10 mM) compounds were added to the compound plate and a 4-fold serial dilution was performed in DMSO. Using an ECHO dispenser, compounds were dispensed at 90 nL/well from the source plate to a 384-well compound plate (PP, low binding). After the dispensing was complete, 30 L/well assay buffer was added to the compound plate and mixed for two minutes on a plate shaker. The cell plate, compound plate, and tips were loaded into the FLIPR instrument, and a transfer of 10 μl of 3× compound to the cell plate was initiated. The treated cell plate was kept at in the dark at 25° C. for 30 minutes. Chloride-free stimulation buffer containing 4× 2 mM Tl+ and 4×EC80 of agonist Loxapine was loaded into a 384-well compound plate (PP, low binding). After the 30-minute incubation, the cell plate, compound plate containing stimulation buffer, and FLIPR tips were loaded into the FLIPR instrument. After a baseline read, the FLIPR initiates a transfer of 10 μL of stimulation buffer containing Loxapine to the cell plate. The plate was read for 160 sec with 1 second interval reads to obtain the data. |
| Affinity data for this assay | |
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