Patch pipettes were pulled from Harvard Apparatus GC150T borosilicate glass (Phymep) by a P-97 puller (Sutter Instrument), coated with SYLGARD, and heat polished. Single-channel currents were amplified with a List LM-EPC7 or a Bio-logic RK 400 patch-clamp amplifier, filtered at 300 or 500 Hz by a LPBF-48DG 8-pole Bessel filter (NPI Electronic), and digitized at a sampling rate of 1–2 kHz using DIGIDATA 1322A or 1440A analogue to digital converters and P-CLAMP software (Axon Instruments) for online monitoring and recording. All experiments were performed at room temperature (22–27°C).
Rk 400
Manufactured by Bio-Logic
Sourced in France
The RK-400 is a multi-purpose laboratory centrifuge designed for a variety of applications. It features a digital display, adjustable speed and time controls, and can accommodate a range of rotor sizes and configurations.
Automatically generated - may contain errors
Lab products found in correlation
Mf 83 microforge, by Narishige (9 mentions)
Pclamp 10, by Molecular Devices (5 mentions)
Dm il inverted microscope, by Leica camera (4 mentions)
Axopatch 200b amplifier, by Molecular Devices (4 mentions)
Pp 83 vertical puller, by Narishige (3 mentions)
P 97 horizontal puller, by Sutter Instruments (3 mentions)
Axopatch 200b, by Molecular Devices (3 mentions)
Axopatch 200b patch amplifier, by Molecular Devices (2 mentions)
Ckx41 inverted microscope, by Olympus (2 mentions)
Pp 830, by Narishige (2 mentions)
Pp 830 vertical puller, by Narishige (2 mentions)
Pp 830 puller, by Narishige (2 mentions)
Digidata 1440a interface, by Molecular Devices (2 mentions)
P 97 puller, by Sutter Instruments (1 mentions)
Pclamp software, by Molecular Devices (1 mentions)
Pp 83, by Narishige (1 mentions)
Ckx41 inverted, by Olympus (1 mentions)
Axoclamp 2b amplifier, by Molecular Devices (1 mentions)
Axoclamp 2b, by Molecular Devices (1 mentions)
Digidata 1400a, by Molecular Devices (1 mentions)
15 protocols using rk 400
1
Patch Clamp Technique for Single-Channel Recording
2
Patch-Clamp Whole-Cell Recordings of GH3 or Jurkat T Cells
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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
3
Whole-Cell Patch-Clamp Technique: Cell Recordings
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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.
4
Patch-Clamp Recordings of GH3 and 13-06-MG Cells
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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.
5
Patch-Clamp Recordings of GH3 and PC12 Cells
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On the day of the experiments, cells (i.e., GH3 or PC12 cells) were harvested and a few drops of cell suspension was immediately transferred to a home-made recording chamber affixed to the stage of a DM-IL inverted microscope (Leica, Wetzlar, Germany). Cells visualized under inverted microscope were immersed at room temperature in normal Tyrode’s solution, the composition of which is detailed above. We performed the patch-clamp recordings under whole-cell configuration with either an RK-400 (Biologic, Echirolles, France) amplifier, or an Axopatch-200B or Axoclamp-2B (Molecular Devices, Sunnyvale, CA, USA) amplifier [20 (link)]. The recording pipette electrodes with tip resistances of 3–5 MΩ were prepared from Kimax-51 glass capillaries (#34500 (outer diameter: 1.5–1.8 mm); Kimble Products, Vineland, NJ, USA), and we fabricated the capillary tubes by using either a PP-83 vertical puller (Narishige, Tokyo, Japan) or a P-97 horizontal puller (Sutter, Novato, CA, USA). An electrode holder filled with a silver chloride-coated silver wire connected the patch electrode to the amplifier. During the measurements, the electrode used was mounted on and delicately controlled by a WR-98 hydraulic micromanipulator (Narishige).
6
Patch-clamp Recordings of Single-channel Currents
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Single-channel currents were recorded from cell-attached or inside-out membrane patches at room temperature using a voltage-clamp amplifier (model RK 400; Bio-Logic, Claix, France). Currents flowing into the pipette were considered to be positive. Channel activity was determined from the average current (I) as NPo = I/i in each patch, where i is the single-channel current, N is the number of open channels, and Po the open-state probability. Currents filtered at 300 Hz were acquired using the pClamp10 software (Molecular Devices) driving an A/D converter (Digidata 1400A, Molecular Devices) at a sampling frequency of 1 kHz.
7
Electrophysiological Recordings of Dissociated Cells
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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.
8
Patch-clamp Recordings in Cultured Cells
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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 IK(DR) recordings with intracellular dialysis, the recording pipettes used were filled with the internal solution containing 0.3 µM CFZ.
9
Patch-Clamp Recordings in Adherent Cells
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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).
10
Patch-clamp electrophysiology on adherent cells
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Before the experiments, we gently dispersed cells with a 1% trypsin/EDTA solution, and an aliquot of cell suspension was directly placed in a recording chamber attached to the fixed-stage of a DM-IL inverted microscope (Leica; Highrise Instrument, Taichung, Taiwan). Cells were immersed at room temperature (20–25 °C) in normal Tyrode’s solution containing 1.8 mM CaCl2. The electrodes that we used were fabricated from Kimax-51 capillaries (Merck, Taipei, Taiwan) using a PP-83 vertical puller (Narishige; Taiwan Instrument, Tainan, Taiwan), and their tips were thereafter fire-polished with an MF-83 microforge (Narishige; Taiwan Instrument, Tainan, Taiwan). As the electrodes were filled with the different internal solutions described above, their resistance was measured to range between 3 and 5 MΩ, for the purpose of avoiding excessive damage to the cell. Patch-clamp recordings were carried out in whole-cell configuration using either an RK-400 (Bio-Logic, Claix, France) or an Axopatch-200B amplifier (Molecular Devices; Bestgen Biotech, New Taipei City, Taiwan), as described elsewhere [31 (link),37 (link),52 (link),67 (link)]. Whole-cell recording was achieved by rupturing the patch of membrane isolated with GΩ sealing by the patch pipet, which brings the cell interior into contact with the pipet interior.
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