All ionic current experiments were performed using excised inside-out patches. Typical current amplitudes were 300–2000 pA at +50 mV test potential, allowing the recording of macroscopic currents. Only those cells with a steady-state current <5% of the peak current were used in the experiments. All measurements were carried out by using Axopatch 200B amplifier connected to a personal computer using Axon Digidata 1550A data acquisition hardware, respectively (Molecular Devices). In general, the holding potential was −100 mV. Experiments were done at room temperature ranging between 20–24 °C. Data were analyzed using the pClamp 10 software package (Molecular Devices). Before analysis, current traces were digitally filtered with a three-point boxcar smoothing filter. A Warner Instruments SF-77A Perfusion Fast-Step system with three-barrel square glass (700 μm internal diameter) was used for rapid solution exchange. This system had a 10–90% exchange time for inside-out patches between 20–30 ms.
Axon digidata 1550a
Manufactured by Molecular Devices
Sourced in United States
The Axon Digidata 1550A is a high-performance data acquisition system designed for electrophysiology experiments. It provides analog-to-digital conversion and signal conditioning for recording a variety of biological signals, including voltage, current, and other physiological measurements.
Automatically generated - may contain errors
Lab products found in correlation
Axopatch 200b amplifier, by Molecular Devices (5 mentions)
Pclamp software, by Molecular Devices (3 mentions)
Axopatch 200b, by Molecular Devices (3 mentions)
Pclamp 10, by Molecular Devices (3 mentions)
Bf150 86 10, by Sutter Instruments (2 mentions)
Axon pclamp 10.7 software suite, by Molecular Devices (2 mentions)
Sf 77a perfusion fast step system, by Warner Instruments (1 mentions)
Axopatch 700b, by Molecular Devices (1 mentions)
Dmi4000b microscope, by Leica Microsystems (1 mentions)
Pm100d, by Thorlabs (1 mentions)
P 1000, by Sutter Instruments (1 mentions)
Clampex 10, by Molecular Devices (1 mentions)
Gc150 10, by Harvard Apparatus (1 mentions)
Borosilicate capillaries, by Sutter Instruments (1 mentions)
Microforge, by Narishige (1 mentions)
Multiclamp 700b, by Molecular Devices (1 mentions)
12 protocols using axon digidata 1550a
1
Macroscopic Ionic Current Experiments
2
Whole-cell Recordings of Membrane Potentials
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Whole-cell recordings were made using TEV-200A amplifiers (Dagan) and digitized by Axon Digidata 1550A (Molecular Devices). The pClamp Software (Molecular Devices) was used for data acquisition at a rate of 5 kHz. Pipettes of 5–20 MΩ resistance were pulled from borosilicate glass and filled with 1 M KCl. Resting and fertilization potentials were generally stable and quantified ∼10 s before and after the depolarization, respectively. Depolarization rates were quantified by determining the maximum velocity of a 1-mV shift in the membrane potential for each recording.
3
Single-Channel Patch-Clamp Recordings
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Single currents were recorded using cell-attached configuration, as described previously [63 (link)]. Briefly, the patch-clamp set-up was based on the Axopatch 200B operational patch-clamp amplifier (Molecular Devices, San Jose, CA, USA) and Axon Digidata 1550A (Molecular Devices, San Jose, CA, USA) analog–digital converter controlled by a Windows-based personal computer with installed Axon PClamp 10.7 Software Suite (Molecular Devices LLC, San Jose, CA, USA) for data acquisition, processing and analysis. Patch pipettes were pulled from borosilicate glass capillaries (BF-150-86-10, Sutter Instruments, Novato, CA, USA) to a resistance of 10–15 MOhm when filled with the extracellular solution containing (in mM): 145 NaCl, 2 CaCl2, 1 MgCl2 and 10 HEPES/TrisOH. Potassium bath solution containing 145 KCl, 2 CaCl2, 1 MgCl2 and 10 HEPES/TrisOH was used to nullify the resting membrane potential of the cells. The pH of all solutions was maintained at 7.3. All experiments were performed at RT. The single-channel recordings were processed and analyzed in Clampfit software (part of Axon PClamp 10.7 Software Suite). Single-channel conductance values were defined by the slope of the current–voltage relationship (I–V) after linear approximation. Averaged conductance values are given as the mean ± SEM (n—number of experiments).
4
Electrophysiology of Xenopus Oocytes
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Electrophysiological recordings were made using TEV-200A amplifiers (Dagan Co.) and digitized by Axon Digidata 1550A (Molecular Devices). Data were acquired with pClamp Software (Molecular Devices) at a rate of 5 kHz. Pipettes used to impale the X. laevis eggs for recordings were pulled from borosilicate glass at a resistance of 5–15 MΩ and filled with 1 M KCl.
5
Whole-Cell Patch-Clamp Voltage Step Recording
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The patch–clamp setup was based on the Axopatch 200B operational amplifier and Axon Digidata 1550A (Molecular Devices LLC, San Jose, CA, USA) analog-to-digital converter, controlled by a Windows-based personal computer with Axon PClamp 10.7 Software Suite (Molecular Devices LLC, San Jose, CA, USA) for data acquisition, filtration, processing and analysis. Pipettes were pulled from borosilicate glass capillaries with filament (BF-150-86-10, Sutter Instruments, Novato, CA, USA) to a resistance of ~5–10 MΩ when filled with a cytosol-like solution (see Section 2.3 ). All experiments were performed at room temperature (22–23 °C). Whole-cell currents were recorded using a whole-cell configuration of the patch–clamp technique, according to the following protocol: current traces were obtained from voltage steps from +20 to +80 mV in 10 mV increments (holding potential was 0 mV); the sampling interval was 1.75 s.
6
Patch-Clamp Recording of Macroscopic Currents
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Standard methods (Hamill et al., 1981 (link)) were used to record currents either in inside-out or outside-out patches. Typical current amplitudes were 300–2,000 pA at +50 mV test potential, allowing the recording of macroscopic currents. Only those cells with a steady-state current <5% of the peak current were in the experiments. Micropipettes were pulled in four stages using a Flaming Brown automatic pipette puller (Sutter Instruments) from Borosilicate Standard Wall with Filament aluminum-silicate glass (Harvard Apparatus) with tip diameters between 0.5 and 1 µm and heat polished to a tip resistance typically ranging from 2 to 8 MΩ. All measurements were performed by using an Axopatch 200B amplifier connected to a personal computer using Axon Digidata 1550A data acquisition hardware, respectively (Molecular Devices). In general, the holding potential was −120 mV. Records were discarded when leak at the holding potential was >10% of the peak current at the test potential. Experiments were done at room temperature (ranging between 20°C and 24°C). Data were analyzed using the pClamp10.5 software package (Molecular Devices). Before analysis, current traces were digitally filtered with a three-point boxcar smoothing filter. Reported errors are SEM.
7
Nanopore-Based Protein Detection Protocol
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Two Ag/AgCl electrodes were inserted into the electrochemical cell filled with electrolyte (1 M KCl, 10 mM Tris-HCl, pH = 8.0). During the detection of protein molecules, the protein molecules were added to the electrolyte solution, with the glass nanopore fixed to the negative electrode and the positive electrode added outside the nanopore (as shown in Figure 1 a). The other ends of the electrodes were connected to the Axopatch 700B patch clamp amplifier (Molecular Devices, San Jose, CA, USA). The signals were digitized using the Axon Digidata 1550A digital-to-analog converter (Molecular Devices, San Jose, CA, USA) and viewed using Clampfit software (Version 10.5.2.6).
8
Whole-cell patch-clamp of cardiomyocytes and HEK cells
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Whole-cell patch-clamp measurements on single isolated cardiomyocytes or HEK 293T cells were performed at room temperature using an inverted DMI 4000B microscope (Leica Microsystems, Wetzlar, Germany), an Axopatch 200B amplifier and an Axon Digidata 1550A (Molecular Devices, San José, CA, United States). Activation light was delivered by a 525-nm LED (Luminus Devices PT-120-G, Sunnyvale, CA, United States) using a 530/20x filter and controlled via custom-built hardware (Essel Research and Development, Toronto, Canada). Light intensity in the object plane was determined with an optical power meter (PM100D, Thorlabs; Newton, NJ, United States) and the illuminated area was determined using a stage micrometer (A = 0.8 mm2). LED-input power was controlled via the custom-built LED control unit. If not specified otherwise, external (bath) and internal (pipette) standard solutions were used (Tables 3 , 4 ).
9
Electrophysiological Characterization of Cells
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A glass pipette was made from a glass capillary (GC150-10, Harvard Apparatus) using a puller (P-1000, Sutter Instrument) to have a pipette resistance of 4–10 MΩ. KCl-based pipette solution was used as the internal solution. For patch-clamp recordings, an amplifier (Axopatch 200B, Molecular Devices) and a digitizer (Axon Digidata 1550A, Molecular Devices) were used. After identifying a cell expressing YC, the cell was contacted and ruptured with a glass pipette and maintained in a whole-cell current clamp mode. Negative current was injected to suppress spontaneous firing. Once the resting membrane potential was stable for > 30 s, command current (300 pA, 5 ms) was injected with a specific frequency (1, 2, 5 and 10 Hz) for 10 s sequentially with a gap of more than 1 min between each frequency. Data was acquired using software (Clampex 10.7, Molecular Devices).
10
Amphotericin B-Perforated Whole-Cell Patch Clamp
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