All patch clamp recordings were performed using a holding potential of −60 mV and currents were acquired with Digidata 1200 board and the pClamp10 software (Axon Instruments, Inc., Foster City, CA, USA). Recording pipettes were pulled from borosilicate glass (WPI, Sarasota, FL, USA) on a horizontal puller (Sutter Instruments, Novato, CA, USA). Patch electrodes were filled with 140 mM KCl, 10 mM BAPTA, 10 mM HEPES (pH 7.4), 4 mM MgCl2, 2 mM ATP and 0.5 mM GTP. Normal external solution for cells was the same as described in the analysis of intracellular Ca2+ transients protocol. For the study of membrane permeability, cell attached configuration was performed adding the different dendrimers in the pipette solution at 1 µM as in perforated patch clamp techniques and solution without dendrimers was used as control. Recordings were performed for 30 min. For synaptic activity evaluation, whole cell configuration was used. Normal external solution was applied by perfusion for two minutes as control and then different dendrimers in normal external solution at 1 µM were applied under the same conditions. In addition, 0.5 µM Tetrodotoxin (TTX) was used to block action potentials.
Horizontal puller
Manufactured by Sutter Instruments
Sourced in Germany
The Horizontal Puller is a laboratory instrument designed for the fabrication of micropipettes and other fine glass structures. It utilizes a controlled pulling motion to draw and shape glass capillary tubing into the desired form.
Automatically generated - may contain errors
Lab products found in correlation
Axopatch 200b amplifier, by Molecular Devices (4 mentions)
Pclamp 10, by Molecular Devices (4 mentions)
Digidata 1200 board, by Molecular Devices (2 mentions)
Multiclamp 700b amplifier, by Molecular Devices (2 mentions)
Eclipse te200 u, by Nikon (1 mentions)
Pclamp 9, by Molecular Devices (1 mentions)
Axopatch 200a amplifier, by Molecular Devices (1 mentions)
Hepes, by Merck Group (1 mentions)
Digidata 1322a, by Molecular Devices (1 mentions)
Borosilicate glass, by World Precision Instruments (1 mentions)
Pclamp software, by Molecular Devices (1 mentions)
Alexa fluor 488 hydrazide, by Thermo Fisher Scientific (1 mentions)
Multiclamp 700b, by Molecular Devices (1 mentions)
Digidata 1550, by Molecular Devices (1 mentions)
Stereotactic frame, by Kopf Instruments (1 mentions)
Forene, by Abbott (1 mentions)
Thin walled borosilicate glass, by World Precision Instruments (1 mentions)
Infusion pump, by KD Scientific (1 mentions)
Oc 725c, by Warner Instruments (1 mentions)
Digidata 1322, by Molecular Devices (1 mentions)
15 protocols using horizontal puller
1
Patch Clamp Recordings of Dendrimer Effects
2
Characterization of Pannexin-1 Channel Properties
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Whole cell membrane current of single oocytes was measured using a two-electrode voltage clamp and recorded with a chart recorder. Both voltage-measuring and current-passing microelectrodes were pulled with a horizontal puller (Sutter Instruments Co) and filled with 3 M KCl. The recording chamber was perfused continuously with frog Ringer (OR2) solution (mM: 82.5 NaCl, 2.5 KCl, 1 CaCl2, 1 MgCl2, 1 Na2HPO4, and 5 HEPES, pH 7.5). Membrane conductance was determined using voltage pulses. Oocytes expressing Panx1were held at −60 mV, and pulses to +60 mV were applied to transiently open the channels by means of the voltage gate. For activation by K+, the membrane potential was held at −50 mV and 10 mV voltage steps were applied at 0.1 Hz for conductance measurements. Voltage ramps were applied with a custom-made device and the ramps lasted 70 seconds.
3
Whole-cell patch-clamp analysis of synaptic activity
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Experiments were performed in the “whole-cell” configuration using the internal and external solutions described below. Synaptic activity was recorded after a stable baseline was reached. Recording pipettes were pulled from borosilicate glass (WPI, Sarasota, FL) in a horizontal puller (Sutter Instruments, Novato, CA). Membrane currents were measured using an Axopatch-200B amplifier (Axon Instruments, Inc., Burlingame, CA) and an inverted microscope (Nikon, Eclipse, TE200-U, Japan). Data was collected, stored and analyzed using a data acquisition system card (Axon Instruments, Inc.) and the pClamp9 software (Axon Instruments, Inc.). For synaptic activity records, data was analyzed using the Minianalysis software, obtaining the frequency, amplitude and decay time of the records. All experiments were performed at room temperature (20–25°C) using a holding potential of −60 mV. Data are given as means ± S.E.M. and are obtained from at least 3 experiments.
4
Patch-Clamp Electrophysiological Recordings of Neuronal Activity
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Electrophysiological recordings were carried out using the patch clamp technique as previously described [8, 37] . Briefly, culture media was changed for an external solution containing (in mM): 150 NaCl, 5.4 KCl, 2.0 CaCl2, 1.0 MgCl2, 10 glucose and 10 HEPES (pH 7.4). The internal solution consisted of (in mM): 120 KCl, 2.0 MgCl2, 2 ATP-Na2, 10 BAPTA, 0.5 GTP, 10 HEPES (pH 7.4). The holding potential was fixed at -60 mV and currents were acquired using a Digidata 1200 board and the pClamp10 software (Axon Instruments, Inc.). Recording pipettes were pulled from borosilicate glass (WPI, Sarasota, FL) on a horizontal puller (Sutter Instruments, Novato, CA) having a resistancebetween 5 and 10 MΩ. Perforated recordings were obtained as previously described [6, 8] . Briefly, Aβ was added to the pipette internal solution and a 5 mV pulse was used to monitor the perforation in cell attached configuration. For evoked current recordings, the experiments were performed at room temperature (20-25°C) using a membrane potential of -60 mV. Data are given as mean±S.E.M. and were obtained from more than 5 experiments. Recordings were performed in the presence of 100 nM TTX (tetrodotoxin) to inhibit action potentials.
5
Whole-cell voltage clamp of HEK-293 cells
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Whole–cell voltage clamp recordings were carried out on transfected HEK-293 cells at a holding potential of −60 mV (at 21 °C) using an Axopatch 200A amplifier (Molecular Devices). Current data were recorded at 10 kHz using a DA/AD interface (Digidata 1322A, Molecular Devices). Patch pipettes were pulled using a horizontal puller (Sutter Instruments) from thin-walled borosilicate glass (Harvard apparatus, Ltd.) that gave resistances between 4 and 7 MΩ when filled with the intracellular electrode solution. The pipette solution comprised of (in mM) 140 K-Gluconate, 1 MgCl2, 4 MgATP, 0.5 EGTA, 10 HEPES, (adjusted to pH 7.3 with KOH, all Sigma). Cells were perfused with the same perfusion buffer used in the calcium flux assay containing 30 µM glycine, and glutamate from 30 nM to 30 µM as detailed in Fig. 3 , using a multichannel perfusion system (Model BPS-8, Scientifica). Transfected cells were identified by an initial large response to maximal glutamate and glycine and used for subsequent recording protocols.
6
ArchT-GFP Expression in PV+ Interneurons
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Injections of viral solution were done under deep anesthesia immediately after intrinsic optical imaging. PV-Cre mice were placed on a warming pad and fixed with a custom-made metal head frame. Three small craniotomies were drilled tangentially around the center of the mapped barrel columns of interest (C1 and C2). Viral solution of rAAV2-FLEX-ArchT-GFP, containing 2 × 1011 viral particles per ml in PBS were delivered by a glass micropipette with microliter graduations (Hirschmann Laborgeräte, Eberstadt, Germany) pulled by a horizontal puller (Sutter Instruments, Novato, CA) and connected to a 50 ml syringe by manual pressure (for details see Fois et al. 43 (link)). Volumes of 750 nl of viral solution were slowly injected within 10 min first at 600 and thereafter at 300 μm cortical depth from the pia. After each injection, the pipette rested in place for another 10 min before slowly retracting. Electrophysiological recordings were performed no earlier than 4 weeks after virus injection. Viral transduction of PV+ interneurons with ArchT-GFP at the probe location was confirmed histologically in all animals (see Fig. 1 A).
7
Artificial Cerebrospinal Fluid Perfusion
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The artificial cerebrospinal fluid (aCSF) contained (in mM): 130 NaCI, 3.5 KCI, 1.25 NaH2PO4, 24 NaHCO3, 2 CaCI2, 1 MgSO4, 10 glucose osmolarity of 300–305 mOsm, equilibrated with 95% O2 and 5% CO2 (pH 7.4). Other agents were added to this medium. In some experiments the aCSF was supplemented with TTX (1 µM) to block voltage-gated Na+ currents. A K+ pipette solution containing (in mM) 130 K-gluconate, 5 KCI, 10 HEPES, 2 MgCI2, 0.5 EGTA, 2 ATP, 1 GTP (pH 7.3) was used in most experiments. Glass micropipettes were pulled with a horizontal puller (Sutter Instruments) using Sutter borosilicate glass capillaries with filament. The electrode resistance after back-filling was 2–4 MΩ. All voltages were corrected for the liquid junction potential (−13 mV).
8
Patch-clamp analysis of IHC mechanoelectric transduction
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Patch-clamp experiments were performed in the standard whole-cell mode using an Axopatch 200B amplifier (Axon Instruments). Patch electrodes were pulled with a horizontal puller (Sutter Ins. Navato, CA) and had a resistance of 2–3 MΩ when filled with pipette solution consisting of (in mM) 135 CsCl, 10 HEPES, 2.5 EGTA, 0.25 CaCl2, MgCl2, 4 MgATP, and 0.4 Na2GTP (pH adjusted to 7.3 with CsOH). The bath solution consisted of (in mM) 130 NaCl, 3 KCl, 1 MgCl2, 10 HEPES, 2.5 CaCl2, and 10 glucose (pH was adjusted to 7.3 using NaOH). Currents were sampled at 20 kHz and filtered at 2 kHz. Voltages were not corrected for a liquid junction potential. No leak current subtraction was performed. Cells were held at −80 mV. All electrophysiological experiments were performed at RT (21-22°C). We used stepwise and sinewave mechanical stimulation of IHC bundles through a piezo-driven fluid-jet stimulator to record IHC mechanoelectrical transducer (MET) currents. We represented the bundle displacement in the form of applied piezo-driven voltage. Bundle displacement was not calibrated for each cell because of variations in stimulating probe positions relative to the stimulated hair bundle.
9
Patch-clamp analysis of ER-derived GUVs
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Isolation of the ER-containing membrane fractions from control and Bcl-2-expressing WEHI7.2 cells and preparation of the GUVs were carried out as described previously [65 (link)]. GUVs were prepared from the 1:5 mixtures of the ER-containing fraction with 10:1 diphytanoylphosphatidylcholine/cholesterol lipid combination (5 mM). The Patch-clamp experiments were carried out using Axopatch 200B amplifier and pClamp 10.0 software (Molecular Devices, Union City, CA) for data acquisition and analysis. Patch pipettes were fabricated from borosilicate glass capillaries (World Precision Instr., Inc., Sarasota, FL) on a horizontal puller (Sutter Instruments Co., Novato, CA) and had a resistance in the range of 7-10 MΩ. Prepared vesicles were immersed in a bath solution containing 150 mM KCl, 10 mM Hepes, 5 mM glucose, pH 7.2. Patch pipettes were filled with the same solution.
10
Electrophysiological Characterization of Spinal Interneurons
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Whole cell patch-clamp recordings were obtained from motoneurons and ventral horn interneurons visualized under infrared-differential interference contrast microscopy. We recorded from a heterogeneous population of interneurons throughout the ventral horn (average whole cell capacitance 32.9 ± 1.7 pF; average input resistance 366.8 ± 34.3 MΩ; n = 119). Interneurons were not readily classifiable into distinct subpopulations based on location, passive properties, or their responses to adenosine. Patch-clamp electrodes (3–5 MΩ) were pulled on a horizontal puller (Sutter Instrument, Novato, CA) from borosilicate glass (World Precision Instruments, Sarasota, FL). Signals were amplified and filtered (4-kHz low-pass Bessel filter) with a MultiClamp 700B amplifier (Molecular Devices) and acquired at ≥10 kHz with a Digidata 1440A A/D board and pCLAMP software (Molecular Devices). Details of voltage- and current-clamp protocols appear in results . We did not correct for the liquid junction potential, which was calculated as 14.2 mV for our solutions (Clampex JPCalcW).
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