Left atrial myocytes were isolated from Etv1f/fMlc2aCre/+ or littermate control (Etv1f/f) hearts that were Langendorff perfused and enzymatically digested for INa recordings as previously described7 (link), 8 (link). All recordings were obtained three times to verify reproducible recordings and within 15 minutes after establishing whole-cell configuration at room temperature with voltage step protocols using an Axon multiclamp 700B Amplifier coupled to a pClamp system (v10.2, Axon Instruments).
Axon multiclamp 700b amplifier
Manufactured by Molecular Devices
Sourced in United States
The Axon Multiclamp 700B amplifier is a versatile laboratory instrument designed for electrophysiological recordings. Its core function is to amplify and condition electrical signals from various types of electrodes, enabling researchers to monitor and analyze the electrical activity of cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Pclamp 10, by Molecular Devices (9 mentions)
Pclamp system, by Molecular Devices (5 mentions)
Pclamp software, by Molecular Devices (4 mentions)
Clampfit 10, by Molecular Devices (4 mentions)
Igor pro, by Wavemetrics (4 mentions)
Digidata 1440a, by Molecular Devices (3 mentions)
Digidata 1440a digitizer, by Molecular Devices (2 mentions)
Axon digidata 1440a, by Molecular Devices (2 mentions)
Matlab, by Wavemetrics (2 mentions)
Axioskop 2 fs plus microscope, by Zeiss (2 mentions)
Cp achromat 10 0.25 air, by Zeiss (2 mentions)
Lumplanfl 60 1.0 na water immersion, by Olympus (2 mentions)
Lb ls 17 lamp, by Sutter Instruments (2 mentions)
Clampex 10, by Molecular Devices (2 mentions)
Patch clamp microscope, by Olympus (1 mentions)
Bx50wi, by Olympus (1 mentions)
Digidata 1440 digitizer, by Molecular Devices (1 mentions)
P 1000 puller, by Sutter Instruments (1 mentions)
Digidata 1440a system, by Molecular Devices (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
44 protocols using axon multiclamp 700b amplifier
1
Isolating Atrial Myocytes for Sodium Current Recordings
2
Patch-clamp analysis of hippocampal neurons
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Whole-cell patch-clamp recordings were performed as previously described [11] . Briefly, primary hippocampal neurons at DIV 9-12 were patched using Axon Multiclamp 700 b amplifier, 1440A Digidata digitizer and pClamp software (Axon Instruments, Foster City, (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted September 11, 2020. ; https://doi.org/10.1101/2020.09.10.291526 doi: bioRxiv preprint 30 CA). Current and voltage recordings were made at 50 kHz, and subsequently filtered at 5 kHz. The recordings were conducted blindly with four groups: untransfected, scrambled LNA Gapmer, ADEPTR Gapmer1, and ADEPTR Gapmer2. Spontaneous excitatory post-synaptic potential currents (sEPSCs) were recorded before and after the bath application of Forskolin under voltage clamp. The current clamp was recorded only to identify the health of the neurons. Only neurons with resting membrane potential of more than -40 mV were used for our analysis.
The copyright holder for this preprint this version posted September 11, 2020. ; https://doi.org/10.1101/2020.09.10.291526 doi: bioRxiv preprint 30 CA). Current and voltage recordings were made at 50 kHz, and subsequently filtered at 5 kHz. The recordings were conducted blindly with four groups: untransfected, scrambled LNA Gapmer, ADEPTR Gapmer1, and ADEPTR Gapmer2. Spontaneous excitatory post-synaptic potential currents (sEPSCs) were recorded before and after the bath application of Forskolin under voltage clamp. The current clamp was recorded only to identify the health of the neurons. Only neurons with resting membrane potential of more than -40 mV were used for our analysis.
3
Sodium Current Characterization in Cardiomyocytes
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All INa recordings in isolated cardiomyocytes were conducted in whole-cell configuration at 25 or 30 °C. Recording pipettes were filled with a solution containing (in mM): NaCl 5, CsF 135, EGTA 10, MgATP 5, HEPES 15, pH 7.2 with CsOH. Cells were maintained in a solution containing (in mM): NaCl 5, CsCl 112.5, TEACl 20, CdCl2 0.1, MgCl2 1, CaCl2, 1, HEPES 20, Glucose 11, pH 7.4 with CsOH. To determine the peak current voltage relation, 200 ms voltage pulses were applied to Vm −90 mV to +30 mV in 5 mV voltage steps, from a holding potential of Vm=−120 mV. Interval between voltage steps was 3 s. Steady state inactivation was determined by stepping Vm to conditioning voltages of between −130 mV and −20 mV for 60 ms, followed by a 30 ms test pulse to Vm=−20 mV to elicit INa. The steady state voltage-dependent inactivation curves were fitted to Boltzmann's functions. As a measure of closed state channel inactivation rate, a 10-sweep protocol used −140 mV hold command followed by depolarization to −50 mV either instantaneously (voltage step) or as a ramp ranging in time from 2 ms (=45 mV ms−1) to 18 ms (=5 mV ms−1). All recordings were obtained utilizing an Axon multiclamp 700B Amplifier coupled to a pClamp system (versions 10.2, Axon Instruments, Foster City, CA).
4
Patch-Clamp Recordings of Neuronal Currents
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Neurons were recorded with Axon MultiClamp 700B amplifier (Axon Instruments, USA) immersed in Tyrode’s solution [12 (link)]. The intracellular solution of glass pipettes (resistance in the range of 3–8 MΩ) contained (in mmol/L): 125 potassium gluconate, 0.5 EGTA, 4 magnesium ATP, 5 NaCl, 0.3 sodium GTP, 10 phosphocreatine, and 10 HEPES (pH 7.2 with KOH). In Supplementary Fig. 3 where voltage clamp was made, intracellular solution consisted of (in mmol/L) 125 Cs-gluconate, 4 magnesium ATP, 0.3 sodium GTP, 10 phosphocreatine, 10 HEPES, 0.5 EGTA, 3.5 QX-314, 5 TEA, and 2 CsCl (pH 7.2 with NaOH). Inward and outward currents were recorded while clamping neurons at −70 and 0 mV, respectively [27 ]. Membrane resistance was measured by injecting a 10-mV step lasting 100 ms in voltage-clamp mode.
5
Patch-Clamp Characterization of Cardiac Sodium Currents
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INa recordings in isolated cardiomyocytes and hiPSC-CM were conducted in whole-cell configuration at room temperature with voltage step protocols as previously described21 (link). As described in published methods21 (link), recording pipettes were filled with a solution containing (in mM) NaCl 5, CsF 135, EGTA 10, MgATP 5, HEPES 15, pH 7.2, with CsOH. Cells were maintained in a solution containing (in mM) NaCl 5, CsCl 112.5, TEACl 20, CdCl2 0.1, MgCl2 1, CaCl2 1, HEPES 20, glucose 11, pH 7.4, with CsOH. All recordings were obtained three times to verify reproducible recordings and within 15 minutes after establishing whole-cell configuration using an Axon multiclamp 700B Amplifier coupled to a pClamp system (version 10.2, Axon Instruments).
6
Electrophysiological Recordings of Spontaneous Action Potentials
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All electrophysiological recordings were conducted at room temperature using an Axonmulticlamp 700B Amplifier and a pClamp system (versions 10.2, Axon Instruments). For spontaneous AP current clamp recordings, pipettes were filled with a solution containing (in mmol/l): KCl 135, MgCl2 1, EGTA 10, HEPES 10, and glucose 5, pH 7.2 with KOH. The bath solution contained (in mmol/l): NaCl 136, KCl 4, CaCl2 1, MgCl2 2, HEPES 10, and glucose 10, pH 7.4 with NaOH. The AP maximum upstroke velocity (dV/dtmax), the maximum negative potentials, action potential amplitudes (APAs), as well as AP durations at 50% (APD50) and 90% (APD90) of repolarization were measured. To avoid the influences of the spontaneous beating rates on the APD, the corrected APD (cAPD) by heart rates [APD/square root of the cycle length between two spontaneous APs (RR)] was used for average and compared between different groups (19 (link)).
7
Patch-Clamp Recordings of Macroscopic Currents
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Macroscopic currents were recorded at room temperature (20–22°C) 1 day after transfection with an Axon MultiClamp 700B amplifier (Axon Instruments). Patch pipettes were pulled to a pipette resistance of 3–5MΩ. The pipette solution (intracellular) contained 140 mM KCl, 2 mM MgCl2, 10 mM EGTA, 10 mM HEPES, and 5 mM Mg-ATP, and the pH was adjusted to 7.4 with KOH. The bath solution contained 138 Mm NaCl, 2 mM CaCl2, 5.4 mMKCl, 1 mM MgCl2, 10 mM d-(+) glucose, and 10 mM HEPES, and the pH was adjusted to 7.4 with NaOH. Current densities were calculated as peak K+ currents at +40 mV. To generate conductance–voltage (G/V) curves, the cells were held at −80 mV and then depolarized for 1.5 s from −80 to +50 mV using an incremental pulse of 10 mV, followed by an isopotential pulse at −10 mV of 400 ms. The current values recorded at the beginning of the−10 mV pulse were normalized and fitted using the Boltzmann function: y = max/[1 + exp(V 0.5 - V)/k], to obtain the half-maximum activation voltage (V0.5) and the slope factor (k).
8
Whole-Cell Sodium Current Measurement
9
Whole-cell patch-clamp recordings of hippocampal neurons
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Whole-cell patch-clamp recordings were performed as previously described (11 (link)). Briefly, primary hippocampal neurons at DIVs 9 to 12 were patched using Axon Multiclamp 700B amplifier, 1440A Digidata digitizer, and pClamp software (Axon Instruments, Foster City, CA). Current and voltage recordings were made at 50 kHz and subsequently filtered at 5 kHz. The recordings were conducted blindly with four groups: untransfected, scrambled LNA Gapmer, ADEPTR Gapmer1, and ADEPTR Gapmer2. sEPSCs were recorded before and after the bath application of forskolin under voltage clamp. The current clamp was recorded only to identify the health of the neurons. Only neurons with resting membrane potential of more than −40 mV were used for our analysis.
10
Electrophysiology of iPSC-derived Neurons
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iPSC-derived neurons were tested on a microscopic workbench with a patch clamp. Neurons were immersed in extracellular fluid containing 95% O2 and 5% CO2 during the whole process. Cells were visualized using an Olympus patch clamp microscope (Olympus Corporation, Tokyo, Japan). Medium-sized neurons with a bright cell margin and smooth surface were selected for patch clamp analysis. Neurons were inserted into the clamp and data were recorded using an Axon MultiClamp 700B amplifier (Axon instruments; Molecular Devices, LLC, Sunnyvale, CA, USA) and Igor 5.0 software (Wavemetrics, Inc., Lake Oswego, OR, USA).
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