| Assay Method Information | |
| | Electrophysiological Assay (EP) (In Vitro Assay) |
| Description: | Sodium currents are measured in the whole-cell configuration using Syncropatch 384PE (Hanlon Technologies, Germany). 1NPC®-384 chips with custom medium resistance and single hole mode are used. Internal solution consists of (in mM): 110CsCl, 10CsCl, 20EGTA, and 10Hepes (pH adjusted to 7.2); and external solution contains (in mM): 60NMDG, 80NaCl, 4 KCl, 1MgCl2, 2CaCl2, 2D-Glucose monohydrate, 10Hepes (pH adjusted to 7.4 with NaOH).After system flushing, testing compounds are dissolved in external solution containing 0.1% Pluronic F-127. The chip is moved into the measuring head and the instrument primes the chip with external and internal solutions. 10 μl cells are added to the chip from a cell hotel, and a negative pressure of −50 mBar is applied to form a seal. Following treatment with seal enhancer solution and wash-off with external solution, negative pressure of −250 mbar is applied for 1 second to achieve the whole-cell configuration, followed by three washing steps in external solution. 20 μl of compounds is added to 40 μl in each well (1:3 dilution of compounds), and after mixing, 20 μl is removed so the volume is retained at 40 ul. After approximately 13 minutes recordings, 20 μl/well of 2 uM TTX, or 333 uM Tetracaine (for Nav1.5) is added to achieve full block. |
| Affinity data for this assay | |
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