| Assay Method Information | |
| | Electrophysiological Assay (EP) (In Vitro Assay) |
| Description: | The following voltage clamp electrophysiology studies are performed on representative compounds using cells heterologously expressing Nav1.7 or Nav1.5 channels. cDNAsfor Nav1.7 (NM_002977) and Nav1.5 (AC137587) are stably expressed in Chinese Hamstr Ovary (CHO) cells and CHL (Chinese Hamster Lung) cells respectively. Sodium currents are measured in the whole-cell configuration using Syncropatch 384PE (Nanlon Technologies, Germany). 1NPC -384 chips with custom medium resistance and single hole mode are used. Internal solution consists of (in mM): 110 CsCl, 10 CsCl, 20 EGTA, and 10 Hepes (pH adjusted to 7.2); and external solution contains (in mM): 60 NMDG, 80 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2), 2 D-Glucose monohydrate, 10 Hepes (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 ul 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 ul of compounds is added to 40 ul in each well (1:3 dilution of compounds), and after mixing, 20 ul is removed so the volume is retained at 40 ul. After approximately 13 minutes recordings, 20 ul/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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