For single-cell electrophysiology recording, cultured neurons were incubated in Tyrode’s buffer containing 20 μM Gabazine, 10 μM NBQX and 25 μM APV. The electrophysiology experiments were performed at room temperature. Borosilicate glass electrodes (Sutter) were pulled to a tip resistance of 2.5–5 MΩ. The glass electrode was filled with internal solution containing 145 mM potassium gluconate, 5 mM MgCl2, 10 mM HEPES, and 4 mM Na2-ATP (pH 7.2, with KOH). The glass electrode’s position was adjusted by a Sutter MP285 micro-manipulator. Membrane potentials were recorded under whole-cell current clamp under I = 0 mode (Axopatch 200B, Axon Instruments). Recorded membrane potential data were filtered with a 5 kHz internal Bessel filter in the amplified and digitized with a National Instruments PCIe-6353 data acquisition 1067 (DAQ) board. The microscope (Nikon Ti-E), the camera (Hamamatsu ORCA-Flash 4.0 v2) and electrophysiology recording system were controlled with a customized software written in LabVIEW 2015 (National Instruments), and the data were extracted and analyzed with a home-made script written in MATLAB R2018b (MathWorks).
Borosilicate glass electrodes
Manufactured by Sutter Instruments
Borosilicate glass electrodes are a type of lab equipment used for various scientific and analytical applications. They are composed of borosilicate glass, which is a durable and heat-resistant material. These electrodes serve as a core component in the measurement and analysis of various parameters, such as pH, ion concentration, and electrical potentials.
Automatically generated - may contain errors
Lab products found in correlation
Mp 285, by Sutter Instruments (3 mentions)
Axopatch 200b, by Molecular Devices (3 mentions)
Orca flash4.0 v2, by Hamamatsu Photonics (2 mentions)
Micropipete puller, by Sutter Instruments (2 mentions)
Axopatch 1d amplifier, by Molecular Devices (2 mentions)
Labview 2015, by National Instruments (1 mentions)
Matlab r2018b, by MathWorks (1 mentions)
Multiclamp 700a amplifier, by Molecular Devices (1 mentions)
Pclamp 10, by Molecular Devices (1 mentions)
Digidata 1440a digitizer, by Molecular Devices (1 mentions)
Matlab, by MathWorks (1 mentions)
Labview, by National Instruments (1 mentions)
Bx50wi, by Olympus (1 mentions)
Clampex, by Molecular Devices (1 mentions)
Multiclamp 700b amplifier, by Molecular Devices (1 mentions)
Pcie 6353, by National Instruments (1 mentions)
7 protocols using borosilicate glass electrodes
1
Single-cell electrophysiology of cultured neurons
2
Whole-cell Recordings of AMPAR-mediated Miniature EPSCs
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Whole-cell recordings in voltage-clamp mode were obtained using borosilicate glass electrodes (Sutter Instrument) with a tip resistance of 3–7 MΩ. The internal solution contained (in mM): 114.5 gluconic acid, 114.5 CsOH, 2 NaCl, 8 CsCl, 10 MOPS, 4 EGTA, 4 MgATP and 0.3 Na2GTP, pH 7.3, adjusted with CsOH.
Signals were amplified with a Multiclamp 700A amplifier, sampled at 20 kHz, filtered at 2.4 kHz, acquired using a Digidata 1440A digitizer and pClamp 10 (all from Molecular Devices). Cells were held at -60 mV, and AMPAR-mediated miniature EPSCS were isolated using bath application of TTX (1 μM), D-AP5 (50 μM) and picrotoxin (100 μM).
ZnCl2 (10 μM) was bath applied in HibernateE to the cultured neurons. Series resistance (Rs) was monitored throughout the duration of all recordings, and data were excluded if Rs increased >20%.
Signals were amplified with a Multiclamp 700A amplifier, sampled at 20 kHz, filtered at 2.4 kHz, acquired using a Digidata 1440A digitizer and pClamp 10 (all from Molecular Devices). Cells were held at -60 mV, and AMPAR-mediated miniature EPSCS were isolated using bath application of TTX (1 μM), D-AP5 (50 μM) and picrotoxin (100 μM).
ZnCl2 (10 μM) was bath applied in HibernateE to the cultured neurons. Series resistance (Rs) was monitored throughout the duration of all recordings, and data were excluded if Rs increased >20%.
3
Measuring GluCl Cl- Conductance in Neuro-2a Cells
4
Electrophysiological Characterization of Rodent DRG Neurons
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Culture and electroporation of rodent DRG neurons were performed as previous described. The borosilicate glass electrodes (Sutter Instrument) were pulled to a tip resistance of 2.5–5 MΩ and filled with internal solution containing 145 mM potassium gluconate, 5 mM MgCl2, 10 mM HEPES, and 4 mM Na2-ATP (pH 7.2, with KOH). The glass electrode’s position was adjusted by a Sutter MP285 micro-manipulator. Membrane potentials were recorded under whole-cell current clamp under I = 0 mode (Axopatch 200B, Axon Instruments). For evaluating intrinsic excitability, DRG neurons were stimulated with stepwise current injection from 5 pA to 100 pA or 110 pA to 300 pA in 20 steps with 473 ms stimulation and 473 ms rest in each step (at 21159.48 Hz) under I-CLAMP NORMAL mode. Recorded membrane potential data were filtered with a 5 kHz internal Bessel filter in the amplified and digitized with a National Instruments PCIe-6353 data acquisition (DAQ) board. The microscope (Nikon Ti-E), the camera (Hamamatsu ORCA-Flash 4.0 v2) and electrophysiology recording system were controlled with a customized software written in LabVIEW (National Instruments), and the data were extracted and analyzed with a home-made software written in MATLAB (MathWorks).
5
Measuring GluCl Cl- Conductance in Neuro-2a Cells
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In order to record GluCl Cl- conductance, under the control
of a human CamKII-α promoter, we used a mouse Neuroblastoma cell line
(Neuro-2a). Heterologous expression of GluCl or eGluCl was obtained with
TurboFect™ transfection reagent (Thermo Fisher Scientific). Whole cell
patch clamp recordings were performed using Borosilicate glass electrodes,
pulled on a micropipete puller (Sutter Instruments) and fire polished
(Narishige), with a final resistance of 2-3.5MΩ. Data were digitized at
5kHz, filtered at 1kHz and recorded with WinEDR software (John Dempster,
Glasgow, UK) and an Axopatch 1-D amplifier (Axon Instruments). Series resistance
compensation of 60% was used throughout. Cells were held at -60mV, and
increasing concentrations of glutamate were applied through a custom-built
single-cell perfusion system. All recordings were performed at room temperature.
The extracellular recording solution contained in mM: 150 NaCl, 2.8 KCl, 2
Ca2Cl, and 10 HEPES, pH adjusted to 7.35 with NaOH, with
glutamate as indicated, and the intracellular recording solution contained: 135
CsCl, 10 Cs-EGTA, 10 HEPES, 1 Mg2Cl, and 4 Na2-ATP, pH
adjusted to 7.35 with CsOH.
of a human CamKII-α promoter, we used a mouse Neuroblastoma cell line
(Neuro-2a). Heterologous expression of GluCl or eGluCl was obtained with
TurboFect™ transfection reagent (Thermo Fisher Scientific). Whole cell
patch clamp recordings were performed using Borosilicate glass electrodes,
pulled on a micropipete puller (Sutter Instruments) and fire polished
(Narishige), with a final resistance of 2-3.5MΩ. Data were digitized at
5kHz, filtered at 1kHz and recorded with WinEDR software (John Dempster,
Glasgow, UK) and an Axopatch 1-D amplifier (Axon Instruments). Series resistance
compensation of 60% was used throughout. Cells were held at -60mV, and
increasing concentrations of glutamate were applied through a custom-built
single-cell perfusion system. All recordings were performed at room temperature.
The extracellular recording solution contained in mM: 150 NaCl, 2.8 KCl, 2
Ca2Cl, and 10 HEPES, pH adjusted to 7.35 with NaOH, with
glutamate as indicated, and the intracellular recording solution contained: 135
CsCl, 10 Cs-EGTA, 10 HEPES, 1 Mg2Cl, and 4 Na2-ATP, pH
adjusted to 7.35 with CsOH.
6
Whole-cell patch-clamp recordings from brain slices
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Coronal brain slices (250 μm) were prepared from 4- to 8-week-old animals for all genotypes tested. The slices were sectioned into ice-cold oxygenated (5% CO2, 95% O2) dissection buffer (in mM: 87 NaCl, 2.5 KCl, 1.6 NaH2PO4, 25 NaHCO3, 75 sucrose, 10 glucose, 1.3 ascorbic acid, 0.5 CaCl2, 7 MgCl2), recovered for 15 min at 37 °C in oxygenated artificial cerebrospinal fluid (aCSF) (in mM: 122 NaCl, 3 KCl, 1.2 NaH2PO4, 26 NaHCO3, 20 glucose, 2 CaCl2, 1 MgCl2, 305–310 mOsm, pH 7.3) and acclimated at room temperature for 10 min before performing electrophysiological recordings. Borosilicate glass electrodes (Sutter Instruments, Novato, CA) were used for whole cell patch clamp recordings. Electrodes were pulled with tip resistance of 2 MΩ, and filled with internal solution (in mM, 120 K-gluconate, 5 KCl, 2 MgCl2, 0.05 EGTA, 10 HEPES, 2 Mg-ATP, 0.4 Mg-GTP, 10 creatine phosphate, 290–300 mOsm, pH 7.3). During recordings, coronal brain slices were placed in a room temperature chamber mounted on an Olympus upright microscope (BX50WI) and perfused with oxygenated aCSF. Cells were visualized under differential interference contrast imaging. Data were obtained via a Multiclamp 700B amplifier, low-pass Bessel-filtered at 4 kHz, and digitized on computer disk (Clampex, Axon Instruments).
7
Single-cell Electrophysiology of Cultured Neurons
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For single-cell electrophysiological recording, cultured neurons were incubated in Tyrode’s buffer containing 20 μM gabazine, 10 μM NBQX, and 25 μM APV (Tyrode’s buffer containing 50 nM 2-APB for HEK293T cells). The electrophysiological experiments were conducted at room temperature. Borosilicate glass electrodes (Sutter) were pulled to a tip resistance of 2.5 to 5 MΩ. The internal solution containing 125 mM potassium gluconate, 8 mM NaCl, 0.6 mM MgCl2, 0.1 mM CaCl2, 1 mM EGTA, 10 mM HEPES, 4 mM Mg-ATP, and 0.4 mM GTP·Na2 (pH 7.3) was adjusted to 295 mOsm/kg with 1 M sucrose and injected into the glass electrode. The glass electrode’s position was adjusted by a Sutter MP285 micromanipulator. An Axopatch 200B (Axon Instruments) amplifier was utilized to clamp the cells. Membrane potential data recorded from the amplifier were filtered by an internal 5-kHz Bessel filter and digitalized at 9681.48 Hz with a National Instruments PCIe-6353 data acquisition (DAQ) board.
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