| Assay Method Information | |
| | hERG Assay |
| Description: | Voltage clamp protocol: Computer software was used to set voltage clamp protocols. In whole-cell model, cells were held at −80 mV, first depolarized to −50 mV for 80 ms, and then depolarized to +20 mV for 4800 ms to activate the hERG channels. After that the cells were repolarized to −50 mV for 5000 ms to elicit the characteristic tail currents. Finally the cells were held at −80 mV again. The peak values of tail currents were sampled for analysis.Automated QPatch procedure: After achieving break-in (whole-cell) configuration, the cells were recorded for 120 sec to assess current stability. The voltage protocol described above was then applied to the cells every 15 sec throughout the whole procedure. Only stable cells with recording parameters above threshold were allowed to enter the drug application procedure. All experiments were conducted at room temperature (about 25° C.). External solution containing 0.1% DMSO (vehicle) was applied to the cells to establish the baseline. After allowing the current to stabilize for 3 minutes, compound was applied. Compound solution was added and the cells were kept in the test solution until the compound's effect reached a steady state or for a maximum of 4 min. For dose response assay, compound was applied to the cells accumulatively from low to high concentrations. Washout with external solution was performed after compound testing. Positive control cisapride is used in the experiments to test the same batch of cells used for test compounds to ensure the normal response and the good quality of the cells.Data were analyzed using Assay Software provided by Sophion, Microsoft Excel and Graphpad Prism. |
| Affinity data for this assay | |
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