Currents were recorded in the whole-cell configuration using a standard patch-clamp technique [42 (link)]. The patch pipettes were pulled from borosilicate glass (ID 1.0 mm and OD 2.0 mm; VITROCOM, Mountain Lakes, NJ, USA) using a micropipette puller (P-97, Sutter Instrument, Novato, CA, USA). The pipettes were filled with an intracellular solution containing (mM) 142 KCl, 5 BAPTA, 5 EGTA and 10 HEPES at pH 7.4. The pipette resistance was 1.5 to 6.0 MΩ. The bath solution contained (mM) 142 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES and 10 glucose at pH 7.4. For control measurements, 142 mM KCl was replaced with 142 mM NaCl in the internal and external solutions. Lifted cells were placed in front of the outlet of the application system with three barrels (inner diameter ~600 µm; Warner Instruments, Hamden, CT, USA) controlled by a step motor (SF-77B, Warner Instruments). Currents were activated by test pulses ranging from −140 to +80 mV in 20 mV increments from a holding potential of 0 mV, with each test pulse lasting 150 ms followed by activation at −60 mV for 100 ms. The time between pulses was 500 ms. Series resistance was compensated for using Patchmaster software up to 80%. At very negative potentials and high expressions of some receptors, a voltage error arose that was not neglectable. The data were not further corrected for this. For ligand jump experiments, 100 µM ATP solutions were applied at a holding potential of −50 mV for one second. The recordings were digitalized using a HEKA EPC 10 (Stuttgard, Germany) amplifier and Patchmaster 2019 software. The sampling rate was 10 kHz and the currents were filtered online at 2.9 kHz using a 4-pole Bessel filter.
Sf 77b
Manufactured by Warner Instruments
Sourced in United States
About the product
The SF-77B is a field stimulator developed by Warner Instruments. It provides precise control over electrical stimulation parameters, including pulse width, amplitude, and frequency. The device is designed for use in electrophysiology and neuroscience research applications.
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106 protocols using sf 77b
1
Whole-Cell Patch-Clamp Recordings of Ionic Currents
2
Electrophysiological Analysis of Autaptic Hippocampal Neurons
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Voltage and current clamp experiments were performed according to our previously published works85 (link) using autaptic hippocampal neurons from P0-P2 Ire1αf/f infected with the control and Cre-encoding lentiviral particles84 (link) produced by the viral core facility of the Charité-Universitätsmedizin Berlin. Whole-cell patch-clamp recordings were performed on the autaptic cultures at room temperature at DIV12-20. Synaptic currents were recorded using a Multiclamp 700B amplifier (Axon Instruments) controlled by Clampex 9.2 software (Molecular Devices). Membrane capacitance and series resistance were compensated by 70%, and data was filtered by a low-pass Bessel filter at 3 kHz and sampled at 10 kHz using an Axon Digidata 1322A digitizer (Molecular Devices). Neurons were perfused (SF-77B, Warner Instruments) with the extracellular solution (in mM): 140 NaCl, 2.4 KCl, 10 HEPES (Merck), 10 glucose (Carl Roth), 2 CaCl2 (Sigma-Aldrich, St. Louis, USA), and 4 MgCl2 (Carl Roth) (~300 mOsm; pH7.4). Somatic patches were carried out using borosilicate glass pipettes, with a tip resistance of 2-3.5 MOhm and filled with the following internal solution (in mM): 136 KCl, 17.8 HEPES, 1 EGTA, 4.6 MgCl2, 4 Na2ATP, 0.3 Na2GTP, 12 creatine phosphate, and 50 U/ml phosphocreatine kinase (~300 mOsm; pH7.4). Data were analyzed using AxoGraph software.
3
Whole-Cell Patch-Clamp Analysis of Autaptic Neurons
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Whole-cell patch-clamp recordings were performed on autaptic cultures at room temperature at days in vitro 13–20 (42 (link)). Synaptic currents were recorded using a Multiclamp 700B amplifier (Axon Instruments) controlled by Clampex 9 software (Molecular Devices). A fast perfusion system (SF-77B; Warner Instruments) continuously perfused the neurons (1 – 2 ml/min) with an extracellular solution that contains the following (in mM): 140 NaCl, 2.4 KCl, 10 HEPES (Merck), 10 glucose (Carl Roth), 2 CaCl2 (Sigma-Aldrich), and 4 MgCl2 (Carl Roth) (~300mOsm; pH 7.4). Somatic whole cell recordings were obtained using borosilicate glass pipettes, with a tip resistance of 2 – 5 MΩ and filled with an intracellular solution that contains the following (in mM): 136 KCl, 17.8 HEPES, 1 EGTA, 4.6 MgCl2, 4 Na2ATP, 0.3 Na2GTP, 12 creatine phosphate, and 50 U/ml phosphocreatine kinase (~300 mOsm; pH7.4). Membrane capacitance and series resistance were compensated at 70% and only neurons with a series resistance lower than 12 MΩ were recorded further. Data were filtered by a low-pass Bessel filter at 3 kHz and sampled at 10 kHz using an Axon Digidata 1322A digitizer (Molecular Devices).
Neurons were clamped at −70 mV, and action potentials triggered by a 2 ms depolarization to 0 mV to measure EPSCs (excitatory postsynaptic currents). To quantify spontaneous release, 40 s of 1 kHz low pass filtered recordings in control and in glutamate receptor antagonist containing solutions were analyzed for the presence of mEPSC events using template algorithm based software in AxoGraph X (AxoGraph Scientific). mEPSCs were defined as events with 0.15 – 1.5 ms rise time and 0.5 – 5 ms half-width. False positive mEPSC events obtained in NBQX were subtracted to calculate the frequency of spontaneous events. The readily-releasable pool (RRP) was determined by applying hypertonic extracellular solution (included additional 500 mM sucrose) for 5 s and integrating the transient inward response component (23 (link)). The Pvr of each cell was calculated by dividing the average charge of the EPSC by the RRP charge. Spontaneous release rate was calculated by dividing the mEPSC frequency by the number of synaptic vesicles in the RRP. The number of synaptic vesicles in the RRP was calculated by dividing the RRP size by the mean mEPSC charge. To measure vesicle fusogenicity, we measured the response onset latency between the open tip control for solution exchange and the onset of the sucrose response (28 (link)).
Neurons were clamped at −70 mV, and action potentials triggered by a 2 ms depolarization to 0 mV to measure EPSCs (excitatory postsynaptic currents). To quantify spontaneous release, 40 s of 1 kHz low pass filtered recordings in control and in glutamate receptor antagonist containing solutions were analyzed for the presence of mEPSC events using template algorithm based software in AxoGraph X (AxoGraph Scientific). mEPSCs were defined as events with 0.15 – 1.5 ms rise time and 0.5 – 5 ms half-width. False positive mEPSC events obtained in NBQX were subtracted to calculate the frequency of spontaneous events. The readily-releasable pool (RRP) was determined by applying hypertonic extracellular solution (included additional 500 mM sucrose) for 5 s and integrating the transient inward response component (23 (link)). The Pvr of each cell was calculated by dividing the average charge of the EPSC by the RRP charge. Spontaneous release rate was calculated by dividing the mEPSC frequency by the number of synaptic vesicles in the RRP. The number of synaptic vesicles in the RRP was calculated by dividing the RRP size by the mean mEPSC charge. To measure vesicle fusogenicity, we measured the response onset latency between the open tip control for solution exchange and the onset of the sucrose response (28 (link)).
4
Rapid Solution Exchange for Amino Acid Signaling
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Fast solution exchanges were performed using the SF-77B (Warner Instruments LLC) piezo-based solution exchange instrument, allowing a time resolution in the 5 to 10 ms range. Amino acid substrate was applied through a theta capillary glass tubing (TG200-4, outer diameter = 2.00 mm, inner diameter = 1.40 mm; Warner Instruments), with the tip of the theta tubing pulled to a diameter of 350 μm and positioned at 0.5 mm to the cell (37 (link)). For paired-pulse experiments, currents were recorded with 10/20/40 ms interval time after removal of amino acid.
5
Whole-cell Patch-clamp Recordings of NMDAR
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Whole-cell patch-clamp recordings were performed 24 h after transfection using fire-polished borosilicate glass pipettes with 3–5 MΩ resistance, filled with internal solution (135 mM CsF, 33 mM CsOH, 2 mM MgCl2, 1 mM CaCl2, 11 mM EGTA, and 10 mM HEPES, adjusted to pH 7.4 with CsOH). The external solution (140 mM NaCl, 2.9 mM KCl, 1 mM CaCl2, and 10 mM HEPES, adjusted to pH 7.4 with NaOH) was locally applied to lifted cells using a stepper motor system (SF-77B, Warner Instruments) in the presence of 1 mM ligands (glutamate and glycine) and in the presence or absence of 30 µM GNE-9278 (Tocris Bioscience, cat. # 6369). Recordings were performed using an Axopatch 200B amplifier (Molecular Devices) at − 60 mV holding potential, acquired at 10 kHz using pCLAMP10.7 software (Molecular Devices), and filtered online at 5 kHz.
6
Patch-clamp recording of P2X receptor currents
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Membrane currents were recorded with a standard patch-clamp technique [30 (link)] in the whole-cell configuration. The patch pipettes were pulled from borosilicate glass (ID 1.0 mm, OD 2.0 mm; VITROCOM, Mountain Lakes, NJ, USA) using a micropipette puller (P-97, Sutter Instrument, Novato, CA, USA). The pipettes were filled with intracellular solution containing (mM) 142 NaCl, 5 BAPTA, 5 EGTA, and 10 HEPES at pH 7.4. The pipette resistance was from 2.0 to 6.0 MΩ. The bath solution contained (mM) 142 NaCl, 10 EGTA, 10 HEPES, and 10 glucose at pH 7.4. To increase the speed of recovery from the hP2X3 receptors, we additionally used a bath solution containing (mM) 142 NaCl, 10 HEPES, 10 glucose, 2 CaCl2, and 1 MgCl2. The cells were lifted for recording from the chamber bottom by the patch pipette and positioned in front of the outlet of the application pipette.
Solutions switches were carried out with the application pipette that had three barrels (inner diameter ~600 µm, Warner Instruments) controlled by a step motor (SF-77B, Warner Instruments). The speed of the laminar solution flow out of the barrels was estimated to be 2–5 cm/s. One barrel contained a control solution and another was connected to a solution selector (Vici Valco Instruments), which allowed us to apply different test solutions by exchanging the solution during the interval (60 sec for P2X2 and P2X2/3 receptors, 180 s for P2X3 receptors) of applying the control solution. Cells expressing hP2X3 were washed with 3 Units/mL apyrase for 30 min to remove ATP. To ensure the comparability of the data points between each application, we rinsed hP2X3 with the following solution for 3 min after each ATP application (mM): 142 NaCl, 10 HEPES, 10 glucose, 2 CaCl2, and 1 MgCl2 at pH 7.4. After sufficient recovery, we switched to the bath solution for 10 s and then to the solution of interest. The speed of the switch around a whole cell was estimated to be below 10 ms by switching between different salt solutions.
The saturation of activation was determined with ATP at 100 µM. The currents were recorded with a HEKA EPC 10 amplifier in combination with the patchmaster software. The sampling rate was 10 kHz and the recordings were on-line filtered at 2.9 kHz using a four-pole Bessel filter. The currents were recorded at a constant holding potential of −50 mV and the series resistance was compensated with the patchmaster software up to 80%.
Solutions switches were carried out with the application pipette that had three barrels (inner diameter ~600 µm, Warner Instruments) controlled by a step motor (SF-77B, Warner Instruments). The speed of the laminar solution flow out of the barrels was estimated to be 2–5 cm/s. One barrel contained a control solution and another was connected to a solution selector (Vici Valco Instruments), which allowed us to apply different test solutions by exchanging the solution during the interval (60 sec for P2X2 and P2X2/3 receptors, 180 s for P2X3 receptors) of applying the control solution. Cells expressing hP2X3 were washed with 3 Units/mL apyrase for 30 min to remove ATP. To ensure the comparability of the data points between each application, we rinsed hP2X3 with the following solution for 3 min after each ATP application (mM): 142 NaCl, 10 HEPES, 10 glucose, 2 CaCl2, and 1 MgCl2 at pH 7.4. After sufficient recovery, we switched to the bath solution for 10 s and then to the solution of interest. The speed of the switch around a whole cell was estimated to be below 10 ms by switching between different salt solutions.
The saturation of activation was determined with ATP at 100 µM. The currents were recorded with a HEKA EPC 10 amplifier in combination with the patchmaster software. The sampling rate was 10 kHz and the recordings were on-line filtered at 2.9 kHz using a four-pole Bessel filter. The currents were recorded at a constant holding potential of −50 mV and the series resistance was compensated with the patchmaster software up to 80%.
7
Whole-cell Patch Clamp Recordings and Analysis
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Whole-cell patch clamp recordings were taken from either neurons or CHO cells in a recording chamber (Corning) mounted onto a fixed-stage upright microscope (Nikon). Patch electrodes (4–6 MΩ) were made from 1.5 mm borosilicate glass (World Precision Instruments). Whole-cell currents were recorded using an EPC-10 patch-clamp amplifier (HEKA). Data were acquired at 10–20 kHz and filtered at 1–3 kHz using a computer equipped with the Pulse 6.0 software (HEKA, Lambrecht). Cells were recorded at a holding potential of −60 mV unless otherwise described. For NMDAR current recordings, the holding potential was −70 mV. A multi-barrel perfusion system (SF-77B, Warner Instruments) and pressure regulator system (ALA-VM8, Scientific Instrument) were used to achieve a rapid exchange of extracellular solutions. The perfusion protocol was set and controlled by the PatchMaster software. To avoid use-dependent desensitization or rundown, ASICs were repeatedly activated by acidic solution at minimal interpulse intervals of >1 min. During each experiment, a voltage step of −10 mV from the holding potential was applied periodically to monitor the cell capacitance and access resistance.
Dose–response curves were fitted to the Hill equation: , where a is the normalized amplitude of the IASICs, b is the concentration of proton in external solution ([H+]), EC50 is the proton concentration or [H+] yielding half of the maximal peak current amplitude and n is the Hill coefficient. The IC50 values for blocker dose–response curves were fitted using the following equation: , where n is the Hill coefficient and IC50 is the concentration of blocker producing 50% of the maximal block (Imax).
Dose–response curves were fitted to the Hill equation: , where a is the normalized amplitude of the IASICs, b is the concentration of proton in external solution ([H+]), EC50 is the proton concentration or [H+] yielding half of the maximal peak current amplitude and n is the Hill coefficient. The IC50 values for blocker dose–response curves were fitted using the following equation: , where n is the Hill coefficient and IC50 is the concentration of blocker producing 50% of the maximal block (Imax).
8
Screening Package for Ion Channel Assay
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Amitriptyline (A-155), bupivacaine hydrochloride (B-125), celecoxib (C-190), citalopram (C-195), flupirtine maleate (F-150), fluoxetine hydrochloride (F-155), ICA-11081 (I-160), L-ascorbic acid (L-140), linopiridine dihydrochloride (L-156), loperamide hydrochloride (L-100), miconazole nitrate (M-206), rosiglitazone (R-125) and 2,5-dimethylcelecoxib (D-150) were selected of a screening package for ion channel from Alomone Labs. BL-1249 (B2186), astemizole (A2861), and DMSO (D2650) were purchased from Sigma-Aldrich. Most stock solutions were prepared in 100% DMSO or water at a concentration of 100 mM. All molecules were diluted in a standard bath solution before starting every experiment. In patch-clamp experiments, drugs were delivered using a perfusion system VC-6 coupled to a fast-step solution charger (SF-77B, Warner Instruments, Hamden, CT, United States).
9
Rapid Spatiotemporal Drug Delivery in Neuroscience
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A three-barrel tube (SF77B, Warner Instruments) was positioned about 200 μm from the brain preparation and controlled by a stepper (SF77B, Warner Instruments). External saline was perfused through the middle barrel to cover the brain preparation, and the drugs in the side barrels were switched to cover the preparations using a step motor, achieved within milliseconds. The timing and duration of drug application were controlled by Clampex and a Digidata 1440A. The following were used: 100 μM CdCl2 (20899, Fluka Sigma-Aldrich)51 (link), 50 μM MCA (M9020, Sigma-Aldrich)51 (link), 2 mM CIM (C4522, Sigma-Aldrich)52 (link), 1 mM histamine (H7250, Sigma-Aldrich)52 (link) and 1 mM ACh (A6625, Sigma-Aldrich)53 (link). Note, Cd2+ is a nonspecific blocker of voltage-gated calcium channels, in addition to some voltage-gated potassium channels. Free calcium concentrations were calculated on the basis of the program provided by https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/CaMgATPEGTA-NIST.htm .
10
Whole-cell ASIC Current Recording Protocol
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Whole-cell ASIC currents were recorded using a combination of patch-clamp and fast perfusion techniques, as described previously [39 (link)]. GFP-positive CHO cells were selected for the recordings of ASIC currents. For fast perfusion, a multibarrel perfusion system (SF-77B, Warner Instruments, Hamden, CT) was used. Patch pipettes were pulled from borosilicate glass. Pipettes had a resistance of 2–4 MΩ when filled with the intracellular solution. Currents were recorded using Axopatch 200B amplifiers (Axon Instruments, Foster City, CA, USA). All data were filtered at 2 kHz and digitized at 5 Hz using Digidata 1320 DAC units (Axon Instruments). Only recordings with an access resistance of less than 10 MΩ and a leak current of less than 100 pA at −60 mV were included for data analysis [40 (link)]. The maximal inward current value was measured as the peak current. The sustained current component of ASIC3 was measured at the end of the 4 sec perfusion of acidic solutions. Since ASIC1a currents show significant run-down in the first ~15 min of whole-cell recording, in general, the effect of KB-R7943 (Sigma-Aldrich, Inc, St. Louis, MO, USA) was tested ~20 min after the initiation of whole-cell configuration and following the recording of at least three stable ASIC currents.
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