Patch-clamp Electrophysiology in GH3 Cells

Shortly before experiments, GH₃ was carefully suspended in normal Tyrode’s solution at room temperature (20–25 °C). A few drops of the suspension containing cell clumps were immediately added to a custom-built chamber on the stage of an inverted Diaphot-200 microscope (Nikon, Tokyo, Japan). Pipettes were pulled from Kimax-51 soft-glass capillaries (#34500-99; Kimble, Vineland, NJ) by using a Narishige PP-830 Vertical Puller (Tokyo, Japan), and their tips were fire-polished using a microforge (MF-83, Narishige). During the measurements, an electrode with a tip resistance of 2–4 MΩ, which was tightly inserted into a holder, was maneuvered using a WR-98 micromanipulator (Narishige). Patch-clamp experiments were performed in the voltage-clamp mode with either cell-attached or whole-cell configuration (rupturing of the membrane patch after GΩ formation) by using a RK-400 Patch-Clamp Amplifier (Bio-Logic, Claix, France) connected to a laptop [36 (link), 44 (link)]. Shortly before GΩ formation, potential correction was performed for a liquid junction potential, which developed at the electrode’s tip because of the difference in the compositions of the internal and bath solutions.