Assay Method Information

Assay Name:  Inhibition Assay
Description:  hERG study was conducted with an automated patch clamp machine, Qpatch-48HT (Sophion Biosciences, Denmark). Cultured CHO cells stably expressing hERG channels (provided by Sophion Biosciences, Denmark) were harvested from culture flasks of 70-90% cell confluence rate and prepared as cell suspension with a cell density of 3-8&timse;10^6 cells/mL in serum-free media (CHO-S-SFM II, cat#12052 Invitrogen; 25 mM HEPES). Cells in such condition were placed into a Qpatch cell stir chamber and used within 4 hours. For each run, cells were first spun down by the built-in Qpatch centrifuge and re-suspended in an extracellular solution (in mM, 2 CaCl2, 1 MgCl2, 4 KCl, 145 NaCl, 10 Glucose, 10 HEPES, pH 7.4, osmolarity ~305 mOsm). Qplate-48 that holds one cell in each of its 48 channels for the later voltage-clamped assay were primed with the extracellular solution and intracellular solution (in mM, 5.4 CaCl2, 1.75 MgCl2, 120 KCl, 10 HEPES, 5 EGTA, 4 NaATP, pH 7.25, Osmolarity ~280-295 mOsm). Cells were dispatched by Qpatch robotic dispensing guns into each Qplate channel and went through the process of giga-ohm sealing and whole cell configuration. Whole-cell recordings were performed in voltage-clamp mode at a holding potential of −80 mV. The hERG current was activated by depolarizing at +20 mV for 5 sec, after which the current was taken back to −50 mV for 5 sec to remove the inactivation and observe the deactivating tail current. The maximum amount of tail current size was used to determine the hERG current amplitude. The above voltage protocol was applied to the cells every 15 sec throughout the whole procedure. External solution containing 0.1% DMSO (vehicle) was applied to the cells to establish the baseline. 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 assays (0.1, 0.3, 1, 3, 10 and 30 μM), the compound was applied to the cells accumulatively from low to high concentrations. Washout with extracellular solution was performed after compound testing. Positive control cisapride 0.1 μM was used on each cell after compound testing to ensure the normal response and the good quality of the cell.
Affinity data for this assay

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