| Assay Method Information | |
| | Qube Assay |
| Description: | Compounds were tested on human NaV1.8 and NaV1.5 channels stably expressed in human embryo kidney (HEK) 293 cells. Sodium current measurements on Qube were conducted as follows: automated 384-well patch-clamp assays on the Qube platform (Sophion Biosciences) were used to measure the inhibition of sodium flow through human NaV1.8 and NaV1.5 channels. Whole-cell voltage-clamp recordings were performed in QChips (Sophion Biosciences) at room temperature. NaV1.8 current measurements on Qube were obtained as follows: NaV1.8 currents were elicited with a 10 second 1 Hertz (Hz) pulse train from a holding potential of −90 millivolts (mV), delivered to the cells once per minute in the control condition (DMSO only) and after compound addition. The 1 hertz pulse train stimulation consisted of ten test pulses to 10 millivolt (mV) for 20 milliseconds (ms), each of which was followed by a 980 millisecond repolarization to −67 millivolts. At the end of the 10 second pulse train stimulation, a 5 second hyperpolarization step to −100 millivolt (mV) was used to recover NaV1.8 from fast inactivation. The peak currents elicited by the 1st and 10th test pulses were used to determine IC50 values for resting inhibition and inactivated state inhibition. NaV1.5 current measurements on Qube were obtained as follows: NaV1.5 currents were elicited with a 20 second 3 Hertz pulse train in the control condition (DMSO only) and after compound addition. The pulse train consisted of sixty 20 millisecond test pulses to 0 millivolt from a holding potential of −80 millivolt (mV). The average peak currents elicited by the last 3 test pulses were used to determine IC50 values for NaV1.5 inhibition. The following buffers were used for the Qube recordings: External buffer for NaV1.8 Qube recording: 150 NaCl, 2 CaCl2, 5 KCl, 1 Mg Cl2, 10 HEPES, 12 Dextrose; External buffer for Qube NaV1.5 recording: 120 N-Methyl-D-Glucamine, 40 NaCl, 1 KCl, 2.7 CaCl2, 5 HEPES, 0.5 MgCl2; and Internal buffer for Qube recording: 120 CsF, 30 CsCl, 10 EGTA, 5 HEPES, 5 NaF, 2 MgCl2. |
| Affinity data for this assay | |
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