Mf 83 microforge

Briefly before the recordings, we harvested GH₃ or Jurkat T cells and rapidly resuspended an aliquot of cell suspension to a custom-made cubicle mounted on the fixed stage of CKX-41 inverted microscope (Olympus; YuanLi, Kaohsiung, Taiwan). We the immersed cells at room temperature (20–25°C) in normal Tyrode’s solution, the composition of which has been described above in detail. We exploited either a P-97 Flaming/Brown horizontal puller (Sutter Instruments, Novato, CA) or a PP-83 vertical puller (Narishige; Taiwan Instrument, Taipei, Taiwan) to fabricate the recording pipette electrodes, which were made of Kimax-51 glass capillaries (Kimble; Dogger, New Taipei City, Taiwan), and we then fire-polished electrode tips with an MF-83 microforge (Narishige). The patch electrodes, in which different internal solutions were filled up, had a tip resistance of 3 to 5 MΩ. In this study, we undertook standard patch-clamp whole cell recordings at room temperature by applying either an RK-400 (Bio-Logic, Claix, France) or an Axopatch-200B patch-amplifier (Molecular Devices, Sunnyvale, CA). To measure whole-cell data, the junctional voltage between the pipette and bath solution was set as zero once the electrode was bathed but shortly before the giga-seal (>1 GΩ) formation. The details of data recordings and analyses achieved in the present work were described in Supplementary Material.

Shortly before each experiment, cells were dissociated, and an aliquot of cell suspension was transferred to a homemade recording chamber positioned on the stage of a CKX-41 inverted microscope (Olympus, Tokyo, Japan). Cells were immersed at room temperature (20–25 °C) in normal Tyrode’s solution containing 1.8 mM CaCl₂. The patch electrodes used were prepared from Kimax capillary tubes (#34500; Kimble Glass, Vineland, NJ, USA) using a vertical two-step electrode puller (PP-83 or PP-830; Narishige, Tokyo, Japan), and their tips were then fire-polished with an MF-83 micro-forge (Narishige). Experiments were performed using the whole-cell configuration of standard patch-clamp technique using either an RK-400 (Bio-Logic, Claix, France) or an Axopatch 200B (Molecular Devices, Sunnyvale, CA, USA) patch-clamp amplifier [19 (link)]. Junctional potentials that developed when the composition of the pipette solution was different from that in the bath were nulled.

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.

Shortly before the experiments, we carefully dissociated cells with a 1% trypsin/EDTA solution, and an aliquot of the suspension containing cell clumps was rapidly placed in a recording chamber adherently attached to the working stage of a DM-IL inverted microscope (Leica; Highrise Instrument, Taichung, Taiwan). The electrodes which were used to record were fabricated from Kimax-51^® capillaries with 1.5–1.8 mm in diameter (Kimble^® 34500-99; Merck, Taipei, Taiwan) by using a PC-10 vertical puller (Narishige; Taiwan Instrument, Tainan, Taiwan), and their tips were then fire-polished with MF-83 microforge (Narishige). When the electrodes were filled up with different internal solutions described above, their resistance was measured to range between 3 and 5 MΩ, for the purpose of making good GΩ-seal formation. We performed patch clamp recordings in cell-attached, inside-out or whole-cell configuration by using either an RK-400 (Bio-Logic, Claix, France) or an Axopatch-200B amplifier (Molecular Devices; Bestgen Biotech, New Taipei City, Taiwan), as elaborated elsewhere [29 (link),31 (link),52 (link),59 (link)]. Whole-cell current recordings were established by rupturing the patch of membrane isolated with GW sealing by the patch pipette, then bringing the cell interior into contact with the pipette interior.

Shortly before the experiments, cells were harvested and an aliquot of cell suspension was transferred to a home-made recording chamber mounted on the fixed stage of CKX-41 inverted microscope (Olympus, Tokyo, Japan). Cells were bathed at room temperature (20–25 °C) in normal Tyrode’s solution, the composition of which was indicated above. The recording electrodes were made of Kimax-51 glass capillaries (Kimble, Vineland, NJ, USA) by using either a PP-83 vertical puller (Narishige, Tokyo, Japan) or a P-97 Flaming/Brown horizontal puller, and their tips were then fire-polished with an MF-83 microforge (Narishige; London, UK). The electrodes used had a resistance of 3–5 MΩ and were filled with different internal solutions as detailed above. Patch-clamp whole cell recordings were performed at room temperature in standard patch-clamp technique by use of either an RK-400 amplifier (Bio-Logic, Claix, France), or an amplifier of Axopatch-200B or Axoclamp-2B (Molecular Devices, Sunnyvale, CA, USA). The junction potential between the pipette and bath solution was nulled after the pipette entered the bath but immediately before seal formation was made, and whole-cell data were hence corrected.

Shortly before the electrical recordings, cells (e.g., GH₃, A7r5, or H9c2 cells) were harvested and transferred to a home-made recording chamber positioned on the stage of an inverted microscope. Cells were immersed at room temperature (22–25 °C) in normal Tyrode’s solution, the composition of which was described above. Patch-clamp recordings under the whole-cell mode were achieved with either an RK-400 (Bio-Logic, Claix, France) or an Axopatch-200B amplifier (Molecular Devices, Sunnyvale, CA) (Wu et al., 2000 (link); Wu et al., 2017 (link)). Patch electrodes with tip resistances of 3–5 MΩ were made of Kimax-51 borosilicate capillaries (#34500; Kimble, Vineland, NJ) on either a PP-830 puller (Narishige, Tokyo, Japan) or a P-97 horizontal puller (Sutter, Novato, CA), and then fire-polished with an MF-83 microforge (Narishige). The signals, comprising voltage and current tracings, were stored online at 10 kHz in an ASUSPRO-BU401LG computer (ASUS, Taipei City, Taiwan) controlled by pCLAMP 10.7 software (Molecular Devices). Changes in membrane potential recorded from GH₃ cells were measured under current-clamp configuration. In a separate set of whole-cell I_K(DR) recordings with intracellular dialysis, the recording pipettes used were filled with the internal solution containing 0.3 µM CFZ.

Shortly before the measurements, cells (e.g., GH₃ or HEK293T cells) were harvested and transferred to a homemade recording chamber positioned on the fixed stage of an inverted Olympus fluorescent microscope (CKX-41; Yuan Yu, Taipei City, Taiwan). Cells were put into in normal Tyrode’s solution at room temperature (22 to 25 °C). After cells were left to adhere to the bottom for several minutes, the recordings were performed. Patch-clamp experiments under either whole-cell, cell-attached, or inside-out mode were achieved with either an RK-400 (Biologic, Claix, France) or an Axopatch-200B amplifier (Molecular Devices, Sunnyvale, CA) [16 (link),35 (link)]. We fabricated patch pipettes, the resistance of which was around 3 to 5 MΩ, from Kimax-51 borosilicate capillaries (#34500; Kimble; Dogger, New Taipei City, Taiwan) pulled on either a PP-830 vertical puller (Narishige, Major Instruments, New Taipei City, Taiwan) or a P-97 programmable horizontal puller (Sutter Instruments, Novato, CA, USA), and the pipettes were then fire polished with an MF-83 microforge (Narishige). During measurements, the digitized signals, consisting of voltage and current tracings, were stored online at 10 kHz in an ASUSPRO-BU401LG computer (ASUS, New Taipei City, Taiwan) controlled by pCLAMP 10.7 software (Molecular Devices).