| Assay Method Information | |
| | Electrophysiology Assay |
| Description: | On the day of experimentation, the 35 mm dish was placed on the stage of an inverted microscope equipped with a perfusion system that continuously perfuses the culture dish with fresh recording media. A gravity driven superfusion system was used to apply test solutions directly to the cell under evaluation. This system consists of an array of glass pipette connected to a motorized horizontal translator. The outlet of the shooter was positioned approximately 100 μm from the cell of interest. Whole cell currents were recorded using the whole-cell patch clamp configuration with the help of an Axopatch 200B amplifier (Axon Instruments, Foster City Calif.), 1322A A/D converter (Axon Instruments) and pClamp software (v. 8; Axon Instruments) and stored on a personal computer. Gigaseals were formed and the whole-cell configuration was established in voltage clamp mode, and membrane currents generated by hNav1.7 were recorded in gap-free mode. Patch clamp pipettes made of borosilicate glass had electrical resistance values between 1.5 and 2.0 MΩ when filled with the pipette solution and series resistance (<5 MΩ) was compensated 75-80%. Signals were sampled at 50 kHz and low pass filtered at 3 kHz. |
| Affinity data for this assay | |
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