Axoclamp 2b amplifier

Manufactured by Molecular Devices

Sourced in United States, United Kingdom, Australia, Israel

The Axoclamp 2B amplifier is a high-performance instrument designed for electrophysiological research applications. It serves as a voltage-clamp and current-clamp amplifier, enabling precise measurements of electrical signals from a variety of biological preparations, such as cells and tissues.

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112 protocols using axoclamp 2b amplifier

Xenopus Oocyte Electrophysiology for P2X7 Receptor

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rP2X₇-encoding RNA was transcribed from pCDNA 3.1x using
the mMessage mMachine T7 Ultra kit. Xenopus oocytes were
injected with 0.5 ng of RNA and incubated at 18 °C for 6 – 12
hours in Barth’s solution plus 250 μg mL⁻¹amikacin. Electrodes (0.5 – 3 MΩ) were filled with 3M KCl,
oocytes were voltage-clamped at −60 mV, and currents were recorded in
buffer containing 100 mM NaCl, 2.5 mM KCl, 0.1 mM EDTA, 0.1 mM flufenamic
acid, and 5 mM HEPES, pH 7.4. Traces were recorded with application of
either 100 μM ATP or 100 μM ATP co-applied with 10 μM
A-438079 antagonist. Data acquisition was performed using the Axoclamp 2B
amplifier and pClamp 10 software (Molecular Devices).
For dose-response curves, traces were recorded with applications of
8 separate ATP concentrations in a dilution series (3 mM, 1 mM, 333
μM, 111 μM, 37 μM, 12 μM, 4 μM, and 1
μM). The maximal amplitude of each current in the series was
normalized as a percentage of the overall maximal current amplitude of the
series. Current-voltage data were recorded with application of 100 μM
ATP by measuring the current during a 1 second voltage ramp from −60
to 60 mV. Data acquisition was performed using the Axoclamp 2B amplifier and
pClamp 10 software (Molecular Devices). Dose-response and current-voltage
experiments were performed in triplicate. Data were averaged and presented
with standard deviation values.

McCarthy A.E., Yoshioka C, & Mansoor S.E. (2019). Full-length P2X7 structures reveal how palmitoylation prevents channel desensitization. Cell, 179(3), 659-670.e13.

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Intracellular Recordings of V1 Neurons

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Intracellular recordings were performed using sharp microelectrodes (borosilicate glass, BF100-50-10; Sutter Instruments, CA) with a resistance of 80-120MO when filled with 3M KCl and 5% Neurobiotin at the tip. The electrodes were lowered into the primary visual cortex using a Sutter P-285 micromanipulator. The intracellular potentials were recorded using an AxoClamp 2B amplifier (Molecular Devices, CA) in the bridge mode, and subsequently low-pass filtered at 3 kHz, digitized at 8–11 kHz (micro1401, Cambridge Electronics Design), and stored on a personal computer (PC) hard drive. V1 neurons were recorded between the depths of 230 and 1100 µM, and cells were later classified into simple and complex types based on spike modulation ratio (Figure 2—figure supplement 1).

Roy A., Osik J.J., Meschede-Krasa B., Alford W.T., Leman D.P, & Van Hooser S.D. (2020). Synaptic and intrinsic mechanisms underlying development of cortical direction selectivity. eLife, 9, e58509.

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Extracellular Recording in Brain Slices

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After sectioning, slices were placed in a Haas-type interface recording chamber and allowed to equilibrate for an hour at the interface between aCSF and moist 95%O₂–5%CO₂ (300 cm³/min). Slices were constantly perfused with aCSF at a flow rate of ∼2 ml/min; the temperature was maintained at 32°C. Slices were visualised with a stereo-microscope (Leica MZ8, Micro Instruments, Long Hanborough, Oxon, UK) mounted above the interface chamber. Extracellular microelectrodes were pulled from thick-walled borosilicate glass capillaries (1.2 mm O.D.×0.69 mm I.D.; Harvard apparatus, Edenbridge, Kent, UK) using a P-97 puller (Sutter Instrument Co, Novato, CA). Electrodes were filled with aCSF and had a typical resistance of 2–4 MΩ. Extracellular potentials were recorded using an Axoclamp 2B amplifier (Molecular Devices, Sunnyvale, CA), low pass Bessel filtered at 1 kHz (NL-125, Digitimer Ltd, Welwyn Garden City, UK) and digitized at 10 kHz by a Power 1401 (CED Ltd, Cambridge, UK). Additionally, a Humbug 50/60 Hz (Digitimer) was used to remove noise locked to the electrical mains supply. Stimulation and data acquisition were controlled using Spike 2 software (v6.12; CED). Data were stored for subsequent off-line analysis using Spike 2.

Powell A.D., Saintot P.P., Gill K.K., Bharathan A., Buck S.C., Morris G., Jiruska P, & Jefferys J.G. (2014). Reduced Gamma Oscillations in a Mouse Model of Intellectual Disability: A Role for Impaired Repetitive Neurotransmission?. PLoS ONE, 9(5), e95871.

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Electrophysiological Recording of Synaptic Plasticity

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Data was acquired with an Axoclamp 2B amplifier (Molecular Devices) by D/A sampling using pCLAMP acquisition software (Molecular Devices). Extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded at 31°C in the CA1 pyramidal cell layer using a glass micropipette filled with ACSF. Schaffer collateral synaptic responses were evoked at 0.033 Hz with a bipolar tungsten stimulating electrode placed near in the stratum radiatum. Stable baseline fEPSPs were confirmed by stimulating at 40–50% maximal field amplitude for 20–30 min prior to beginning experiments. Baseline fEPSPs were recorded at 40–50% of maximal amplitude for 15 min by stimulating every 30 sec. Train fEPSPs were recorded for an additional 60 min. LTP was generated with two high-frequency trains of 1 s each at 100 Hz, 20 s apart, using the maximal stimulus intensity. We generated LTD using a 15 min 1 Hz train at the half-maximal intensitiy. The mean initial slopes (between the 0 and 50% points on the rising phase) of two averaged fEPSPs were compared between treatment groups.

Lykens N.M., Coughlin D.J., Reddi J.M., Lutz G.J, & Tallent M.K. (2017). AMPA GluA1-flip targeted oligonucleotide therapy reduces neonatal seizures and hyperexcitability. PLoS ONE, 12(2), e0171538.

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Hippocampal Long-Term Potentiation Induction

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Mouse brains were subjected for horizontal sectioning, and subjected to extracellular recordings of field excitatory postsynaptic potentials (fEPSPs) using a recording electrode in the CA1 region of the hippocampus and a stimulating electrode in the CA3 region. The baseline of fEPSP was monitored, and then high-frequency stimulation (HFS) at 100Hz was applied. Evoked fEPSPs were recorded via an AxoClamp-2B amplifier (Molecular Devices), and analyzed using pClamp 10.5 software (Molecular Devices). The long-term potentiation (LTP) of CA1 was calculated via the averaged changes of fEPSP slopes at 0 and 40 minutes after HFS.

Her L.S., Mao S.H., Chang C.Y., Cheng P.H., Chang Y.F., Yang H.I., Chen C.M, & Yang S.H. (2017). miR-196a Enhances Neuronal Morphology through Suppressing RANBP10 to Provide Neuroprotection in Huntington's Disease. Theranostics, 7(9), 2452-2462.

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Electrophysiology of Drosophila Neuromuscular Junction

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Wandering third instar larvae were dissected in cold HL3 solution following standard protocol [80 (link)]. The spontaneous (mEJC) and evoked (EJC) membrane currents were recorded from muscle 6 in abdominal segment A3 with standard two-electrode voltage-clamp technique [41 (link)]. All the recordings were performed at room temperature in HL3 solution containing 0.5mM Ca2+ unless otherwise indicated. The current recordings were collected with AxoClamp2B amplifier (Molecular Devices Inc.) using Clampex 9.2 software (Molecular Devices Inc.). The nerve stimulation was delivered through a suction electrode which held the cut nerve terminal cord. In all voltage clamp recordings, muscles were held at -80 mV. The holding current was less than 5 nA for 90% of the recordings and we rejected any recording that required more than 10 nA current to maintain the holding potential.
The amplitudes of mEJC and EJC were measured using Mini Analysis 6.0.3 software (Synaptosoft) and verified by eye. QC was calculated by dividing the mean EJC amplitude by mean mEJC amplitude. The recording traces were generated with Origin 7.5 software (Origin Lab).Spontaneous and evoked potentials were measured as previously described [49 (link)]. Standard two-electrode voltage-clamp technique was used as described in [44 (link)].

Liao E.H., Gray L., Tsurudome K., El-Mounzer W., Elazzouzi F., Baim C., Farzin S., Calderon M.R., Kauwe G, & Haghighi A.P. (2018). Kinesin Khc-73/KIF13B modulates retrograde BMP signaling by influencing endosomal dynamics at the Drosophila neuromuscular junction. PLoS Genetics, 14(1), e1007184.

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Expression and Characterization of Asu-acr-16 Receptor

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Full length cRNA of Asu-acr-16 and the ancillary gene, Asu-ric-3 (UniProtKB accession number: F1L1D9_ASCSU). were prepared using the previously described methods (Zheng et al., 2016 ). A cRNA mixture of 25 ng Asu-acr-16 and 5 ng Asu-ric-3 cRNA in 50 nL RNAse-free water was injected into de-folliculated Xenopus laevis oocytes (Ecocyte Bioscience, Austin, TX, USA). The injected oocytes were incubated in incubation solution (100 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂·2H₂O, 1 mM MgCl₂·6H₂O, 5 mM HEPES, 2.5 mM Na pyruvate, 100 U/mL penicillin, 100 μg/mL streptomycin, pH 7.5) at 19 °C for 4–8 days, with 100 μM BAPTA-AM added ∼3 h before recording.
A two-electrode voltage-clamp technique was used to record currents from the Asu-ACR-16 receptor expressed in the Xenopus oocytes. The oocytes were kept in recording solution (100 mM NaCl, 2.5 mM KCl, 1 mM CaCl₂·2H₂O and 5 mM HEPES, pH 7.3) and clamped to −60 mV. Inward currents were induced by addition of chemicals that acted as agonists that opened the nicotinic ion-channel receptors. An Axoclamp 2B amplifier (Molecular Devices, CA, and USA) was used to record the currents that were acquired with Clampex 9.2 (Molecular Devices, CA, USA) software and analyzed using GraphPad Prism 5.0 (GraphPad Software Inc. CA, USA).

Zheng F., Du X., Chou T.H., Robertson A.P., Yu E.W., VanVeller B, & Martin R.J. (2016). (S)-5-ethynyl-anabasine, a novel compound, is a more potent agonist than other nicotine alkaloids on the nematode Asu-ACR-16 receptor. International Journal for Parasitology: Drugs and Drug Resistance, 7(1), 12-22.

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Intracellular Recordings in 6-OHDA-Lesioned Rats

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After 3 weeks of recovery, we performed intracellular recordings in 6-OHDA-lesioned and sham rats maintained in narcotized and sedated states with fentanyl (4 µg/kg, i.p.; Janssen-Cilag, Issy-Les-Moulineaux, France), immobilized with gallamine triethiodide (40 mg, i.m., every 2 h; Specia, Paris, France) and artificially ventilated (UMV-03, UNO, Zevenaar, Netherlands). Craniotomies were drilled above M1 (from the interaural line: AP 12.5 mm; ML 3.8 mm) and STN (AP 5.2 mm; ML 2.5 mm). Body temperature was maintained at 36.5 °C with a homeothermic blanket. Intracellular recordings were performed using glass micropipettes filled with 2 M potassium acetate (40–70 MΩ) and the active bridge mode of an Axoclamp 2B amplifier (Molecular Devices, Union City, CA). Data were sampled at 25 kHz via a CED 1401 interface using the Spike2 data acquisition program (Cambridge Electronic Design, Cambridge, UK). The STN ipsilateral to the recorded pyramidal cells was stimulated with a bipolar electrode (SNE-100; Rhodes Medical Instruments, Woodlands Hill, CA) at 8.1 mm depth. Electrical stimulation consisted in either single-shock STN stimulations or 130 Hz DBS (60 µs, 2–4 V), using a stimulus isolator (DS2A, Digitimer, WelWyn Garden City, UK) driven by a pulse stimulator (Pulsemaster A300, WPI, Hitchin, UK).

Valverde S., Vandecasteele M., Piette C., Derousseaux W., Gangarossa G., Aristieta Arbelaiz A., Touboul J., Degos B, & Venance L. (2020). Deep brain stimulation-guided optogenetic rescue of parkinsonian symptoms. Nature Communications, 11, 2388.

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Cardiac Electrophysiology Recording Protocol

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Sharp microelectrodes (20–40 MΩ) were used to record action potentials, Na⁺/Ca²⁺ exchange (I_NCX) and L-type Ca²⁺ channel (I_Ca,L) currents, using a switch-clamping system (Axoclamp 2B Amplifier, Molecular Devices, LLC, San Jose, California). Late Na⁺ (I_Na,Late) and Na⁺/K⁺ ATPase currents were measured in the whole-cell configuration using patch pipettes (with resistances 4–7 MΩ when filled with their pipette solutions). The protocols for these electrophysiological measurements are detailed in Ke at al. (14 (link)). During the electrophysiological experiments, myocytes were superfused with normal Tyrode's solution at 37°C, except during the recordings of I_Na,Late, which were at room temperature. I_Na,Late was evaluated as ranolazine-sensitive current.

Firth J.M., Yang H.Y., Francis A.J., Islam N, & MacLeod K.T. (2020). The Effect of Estrogen on Intracellular Ca2+ and Na+ Regulation in Heart Failure. JACC: Basic to Translational Science, 5(9), 901-912.

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Patch-Clamp Recordings of GH3 and 13-06-MG Cells

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GH₃ or 13-06-MG cells were harvested and rapidly transferred to a customized chamber shortly before the electrical recordings. The chamber was positioned on the stage of an inverted microscope. Cells were kept for immersion in normal Tyrode’s solution at 20–25 °C; the composition of this solution is described above. Patch-clamp recordings were undertaken under whole-cell mode with either an RK-400 (Biologic, Echirolles, France) or an AxoClamp 2B amplifier (Molecular Devices; Kim Forest, Tainan, Taiwan) [52 (link),53 (link)]. Patch electrodes with tip resistance of 3–5 MΩ were made from Kimax-51 capillaries (#34500 (1.5–1.8 mm in outer diameter); Dogger, Tainan, Taiwan), using either a PP-830 vertical puller (Narishige, Tokyo, Japan) or a P-97 horizontal puller (Sutter, Novato, CA), and their tips were then fire-polished with MF-83 microforge (Narishige). The signals, which comprised voltages and current tracings, were stored online at 10 kHz in a touchscreen computer (ASUSPRO-BU401LG, ASUS, Tainan, Taiwan) equipped with Digidata 1440A interface (Molecular Devices), controlled by pCLAMP 10.7 software (Molecular Devices). The potentials were revised for the liquid–liquid junction potential that appeared when the composition of the pipette solution was different from the solution of the bath.

Hsiao H.T., Lu G.L., Liu Y.C, & Wu S.N. (2021). Effective Perturbations of the Amplitude, Gating, and Hysteresis of IK(DR) Caused by PT-2385, an HIF-2α Inhibitor. Membranes, 11(8), 636.

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