Pp 830 vertical puller

GH₃ or 13-06-MG cells were harvested and rapidly transferred to a customized chamber shortly before the electrical recordings. The chamber was positioned on the stage of an inverted microscope. Cells were kept for immersion in normal Tyrode’s solution at 20–25 °C; the composition of this solution is described above. Patch-clamp recordings were undertaken under whole-cell mode with either an RK-400 (Biologic, Echirolles, France) or an AxoClamp 2B amplifier (Molecular Devices; Kim Forest, Tainan, Taiwan) [52 (link),53 (link)]. Patch electrodes with tip resistance of 3–5 MΩ were made from Kimax-51 capillaries (#34500 (1.5–1.8 mm in outer diameter); Dogger, Tainan, Taiwan), using either a PP-830 vertical puller (Narishige, Tokyo, Japan) or a P-97 horizontal puller (Sutter, Novato, CA), and their tips were then fire-polished with MF-83 microforge (Narishige). The signals, which comprised voltages and current tracings, were stored online at 10 kHz in a touchscreen computer (ASUSPRO-BU401LG, ASUS, Tainan, Taiwan) equipped with Digidata 1440A interface (Molecular Devices), controlled by pCLAMP 10.7 software (Molecular Devices). The potentials were revised for the liquid–liquid junction potential that appeared when the composition of the pipette solution was different from the solution of the bath.

Prior to each experiment, cells (i.e., GH₃ cells and mHippoE-14 neurons) were gently dissociated, and we transferred an aliquot of cell suspension to a home-made chamber mounted on the fixed stage of an inverted TMS-F microscope (Nikon, Tokyo, Japan). The microscope was coupled to a video camer system with magnification up to 1500×. Cells were immersed at room temperature (20–25 °C) in normal Tyrode’s solution. The patch electrodes used were drawn from Kimax-51 glass capillaries (#34500; Kimble, Vineland, NJ, USA) on a PP-830 vertical puller (Narishige, Tokyo, Japan), and their tips fire-polished with MF-83 microforge (Narishige). When the electrodes were filled with different internal solutions, their resistances generally ranged from 3 to 5 MΩ. A three-dimensional oil-driven micromanipulator (MO-103; Narishige, Tokyo, Japan) was used to precisely position the electrode near the cell examined. Ionic currents were measured in the whole-cell, cell-attached or inside-out configuration of the patch-clamp technique by using an RK-400 patch-clamp amplifier (Bio-Logic, Claix, France) [51 (link),52 (link)]. The liquid junction potentials were generally nulled shortly before seal formation was made, and the whole-cell results were corrected by the potentials measured under our experimental conditions.

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.