Stretch-activated currents were recorded using Axopatch 200B amplifier (Molecular Devices Axopatch 200B). Currents were sampled at 20 kHz and filtered at 2 kHz. External solution used to zero the membrane potential consisted of (in mM) 140 KCl, 1 MgCl2, 10 glucose, and 10 HEPES (pH 7.3 with KOH). Recording pipettes were of 2–3 MΩ resistance when filled with standard solution composed of (in mM) 130 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 TEA-Cl, and 10 HEPES (pH 7.3 with NaOH). When specified, pipette solution was supplemented with 30 μM Yoda1. Membrane patches were stimulated with 500-ms negative pressure pulses through the recording electrode using Clampex controlled pressure clamp HSPC-1 device (ALA-Scientific, Farmingdale NY). Consecutive sweeps with pressure stimulation ranging from 0 to −80 mm Hg (Δ-10 mm Hg) were applied every 15 s. Full-trace histograms in Figure 2D were fitted with Gaussian equations using multi-peak fitting analysis of IGOR Pro software.
Axopatch 200b
Manufactured by Molecular Devices
Sourced in United States, United Kingdom, France
The Axopatch 200B is a high-performance patch-clamp amplifier designed for electrophysiological research. It is capable of recording and amplifying small electrical signals from cells and tissues with high precision and low noise.
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497 protocols using axopatch 200b
1
Stretch-Activated Ion Channel Recordings
2
Electrophysiological Characterization of DMD-hPSC-derived CCs
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Electrophysiological recordings were obtained using whole-cell patch-clamp technique on an Axopatch 200B (Axon Instruments, Foster City, CA) at room temperature (22–24°C). The recording pipettes contained: 120 mM KCl, 8 mM EGTA, 10 mM HEPES, 6.8 mM MgCl2, 3 mM CaCl2, 4 mM ATPNa2, 0.4 mM GTPNa2 (pH 7.2). The bath solution contained: 130 mmol/L NaCl, 4 mM/L KCl, 1.8 mM/L MgCl2, 1.8 mM/L CaCl2, 10 mM/L HEPES, 11 mM/L glucose (pH 7.4). Action potentials (APs) were measured in the current-clamp configuration in response to brief (1–2 ms) depolarizing current injections at 1 Hz. The resting membrane potential, the amplitude and duration (APDs) at 20, 50, and 90% repolarization of the APs were measured (respectively indicated as APD20, APD50, and APD90). 7 DMD-hPSC-derived CCs (DMD-CCs) and 18 WT-hPSC-derived CCs (WT-CCs) were analyzed. Cells arrhythmogenicity was tested using application of successive 10 s runs in the current clamp configuration defined by 5 brief (1 ms) depolarizing current injections at 2 Hz followed by a resting period. Data were analyzed using the Clampfit 10.0 (Axon Instruments) software.
3
Voltage-Gated Calcium Channel Characterization
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Whole-cell currents in the A7r5 cells were recorded using an Axopatch 200B or 700B amplifier (Axon Instruments). The bath solution contained (in mM): 143 TEACl, 10 BaCl 2 , 2 MgCl 2 , 10 HEPES, 10 glucose, pH 7.4 (adjusted with TEAOH). The internal solution contained (in mM): 125 CsCl, 10 HEPES, 10 EGTA, 1 MgCl 2 , 1 CaCl 2 , 3 Mg-ATP, 0.3 Tris-GTP (pH adjusted to 7.2 using CsOH). The pipettes were created from capillary tubing (BRAND, Wertheim, Germany) and had resistances of 4-6 MΩ under these solution conditions. All of the recordings were performed at room temperature. Steady-state calcium channel activation was determined by peak currents using the following protocol. The cells were held at -50 mV and depolarized in 10-mV steps from -70 to +10 mV at 10-s intervals upon repolarization to -50 mV. For steady-state channel inactivation, the cells were tested at +10 mV from a pre-pulse incremented in 10 mV (-90 mV to +50 mV; 10 s) upon repolarization to -90 mV. The currents were sampled at 10 kHz and filtered at 2 kHz, and corrected online for leak and residual capacitance transients using a P/4 protocol.
4
Quantitative Analysis of Ion Channel Unitary Currents
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Electric potentials expressed as trans relative to cis at virtual ground. Control of the bilayer potential and recording of unitary currents was done using an Axopatch 200B amplifier (Axon Instruments Pty, Ltd). Channel currents were digitized at 50 kHz and low pass filtered at 5 kHz. Before analysis the current signal was re-digitized at 1 kHz and low pass-filtered at 500 Hz. Channel substate analysis was carried out using the Hidden Markov Model (HMM) 46 . The algorithm calculates from the raw signal the most likely amplitude histogram, the idealised, three-level, current time course with background noise subtracted and the transition-rate matrix using maximum likelihood criteria.
5
Patch-Clamp Analysis of VSMC Membrane Potential
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The membrane potential in VSMCs was recorded as previously described (Lee et al. 2020) (link). Briefly, the membrane potential of VSMCs was measured using a whole-cell patch-clamp technique in current-clamp mode (I = 0). The membrane potential was amplified using a patch-clamp amplifier (Axopatch 200B; Axon Instruments, Union City, CA, USA). The bath solution (pH 7.4) consisted of 135 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 5 mM glucose, and 10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES). The pipette solution (pH 7.3) contained 150 mM KCl, 1 mM MgCl 2 , 5 mM EGTA, and 10 mM HEPES. The resistance of the pipette tip was 4-6 MΩ. The chemicals were dissolved in the bath solution at the desired concentrations. The experimental data were analyzed using Clampfit (pCLAMP, version 9.2, Axon Instruments).
6
Patch-clamp Recordings with Pressure Clamp
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Patch-clamp recordings were carried out as we described previously (Fatehi et al., 2017) (link), under voltage clamp at room temperature (~22°C). The recording pipette and chamber were coupled by Ag/AgCl electrodes to an Axopatch 200B patch-clamp amplifier and Digidata 1200A BNC dataacquisition system, controlled by pCLAMP 10 software (Axon Instruments). Both bath and pipette solutions were composed of (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, 10 glucose and pH 7.4. The seal resistance was no less than 5 GΩ for all cell-attached recordings.
The recordings were low-pass filtered at 1 kHz following acquisition. Linear stepwise negative pressures (by suction) of various magnitudes were applied to the interior of the glass pipette using a high-speed pressure clamp system (HSPC-1, ALA Scientific, Farmingdale, NY).
The recordings were low-pass filtered at 1 kHz following acquisition. Linear stepwise negative pressures (by suction) of various magnitudes were applied to the interior of the glass pipette using a high-speed pressure clamp system (HSPC-1, ALA Scientific, Farmingdale, NY).
7
Patch-clamp Electrophysiology Technique
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The cells were transferred to a bath mounted on the stage of an inverted microscope (TE20000-S;
(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 October 21, 2020. ; https://doi.org/10.1101/2020.10.20.346940 doi: bioRxiv preprint Nikon, Tokyo, Japan). The bath (0.15 ml) was continuously perfused at 5 ml•min -1 . Borosilicated glass pipettes with a free-tip resistance of ~2.5 MΩ were connected to the CV 203Bu head stage of a patchclamp amplifier (Axopatch 200B; Axon Instruments, San Jose, CA). The series resistance, estimated by dividing the time constants of capacitive current, was kept below 10 MΩ in the whole-cell configuration.
To correct the cell size, the current amplitudes were divided by cell capacitance and expressed as pA•pF - 1 . The pipettes were pCLAMP software version 10.6.2 and Digidata-1440A (Axon Instruments) were used to acquire data and apply command pulses. The recorded currents were sampled at 10 kHz and were lowpass Bessel-filtered at 5 kHz.
(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 October 21, 2020. ; https://doi.org/10.1101/2020.10.20.346940 doi: bioRxiv preprint Nikon, Tokyo, Japan). The bath (0.15 ml) was continuously perfused at 5 ml•min -1 . Borosilicated glass pipettes with a free-tip resistance of ~2.5 MΩ were connected to the CV 203Bu head stage of a patchclamp amplifier (Axopatch 200B; Axon Instruments, San Jose, CA). The series resistance, estimated by dividing the time constants of capacitive current, was kept below 10 MΩ in the whole-cell configuration.
To correct the cell size, the current amplitudes were divided by cell capacitance and expressed as pA•pF - 1 . The pipettes were pCLAMP software version 10.6.2 and Digidata-1440A (Axon Instruments) were used to acquire data and apply command pulses. The recorded currents were sampled at 10 kHz and were lowpass Bessel-filtered at 5 kHz.
8
Optical Fiber Temperature Measurement
9
Graphene Device Ion Channel Characterization
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Ion channel activity was measured by a patch clamp amplifier (Axopatch 200B, Axon Instruments), which was placed on a vibration isolation table with a Faraday cage shield. The electrolyte voltage was applied by a Ag/AgCl electrode, and ground was connected to the source terminal of the graphene device, with the drain terminal floating. The signal was acquired and digitized by Digidata 1440A (Axon Instrument) and passed through a 1 kHz filter and digitized at a 10 kHz sampling rate. Data collection was performed by electrophysiology software (pClamp10).
10
Isolation and Characterization of Pulmonary Artery Smooth Muscle Cells
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PASMC were isolated as previously described [4 (link),5 (link)]. Membrane currents were recorded with an Axopatch 200B and a Digidata 1322A (Axon Instruments, Burlingame, CA, USA) using the whole-cell configuration of the patch-clamp technique. Myocytes were superfused with an external Ca2+-free Hepes solution containing (in mM): NaCl 130, KCl 5, HEPES 10, MgCl2 1.2 and glucose 10 (pH adjusted to 7.3 with NaOH) and a Ca2+-free pipette (internal) solution containing (in mmol/L): KCl 110, MgCl2 1.2, Na2ATP 5, HEPES 10, EGTA 10 (pH adjusted to 7.3 with KOH). Total K+ current was evoked following the application of 250 ms depolarizing pulses from −60 mV to +60 mV in 10 mV increments. To characterize TASK currents, cells were clamped at 0 mV for 3 min, allowing Kv current inactivation and isolating the non-inactivating current, IKN. Thereafter, a 1 s voltage ramp from +60 to −100 mV was applied. The ramp was applied again after 5 min perfusion with an external solution buffered at pH 6.3. Non-inactivating TASK currents were identified as the current sensitive to pH [24 (link)]. Currents were normalized to cell capacitance and expressed in pA/pF. Membrane potential was recorded under the current-clamp mode. All experiments were performed at room temperature (22–24 °C).
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