Wr 98 micromanipulator

Shortly before each experiment, cells (i.e., GH₃ or INS-1 cells) were dissociated and a few drops of cell suspension was transferred to a home-made chamber mounted on the fixed stage of an inverted Diaphot-200 microscope (Nikon, Tokyo, Japan). They were immersed at room temperature (20–25 °C) in normal Tyrode’s solution, the composition of which is described above. We fabricated the recording electrode from Kimax-51 glass capillaries (#34500; Kimble, Vineland, NJ) using a PP-830 vertical puller (Narishige, Tokyo, Japan) in which a two-step pull mechanism was applied, and their tips were fire-polished with a microforge (MF-83, Narishige). During the measurements, the electrode with tip resistance ranging from 3 to 5 MΩ, which was firmly inserted into holder, was maneuvered by use of a WR-98 micromanipulator (Narishige). Patch-clamp experiments operated under voltage- or clamp-clamp mode were carried out by using an RK-400 patch-clamp amplifier (Bio-Logic, Claix, France) connected with a personal computer [43 (link)]. Shortly before giga-seal formation was achieved, the potentials were commonly corrected for the liquid junction potential that generally developed at the pipette tip, as the composition of internal solution was different from that in the bath.

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.