Whole-cell patch-clamp recordings were based on our previous studies.18 ,20 (link) Experiments were performed in a recording chamber on the stage of an Olympus BX51W1 microscope equipped with infrared differential interference contrast optics for visualization. Excitatory postsynaptic currents (EPSCs) were recorded in the voltage-clamp configuration, and the resting membrane potential was held at −60 mV from layer II/III pyramidal neurons in the ACC with an Axon 200B amplifier (Axon Instruments). The recording pipettes (3–5 MΩ) were filled with a solution containing (in mM) 145 K-gluconate, 5 NaCl, 1 MgCl2, 0.2 EGTA, 10 HEPES, 2 Mg-ATP, and 0.1 Na3-GTP (adjusted to pH 7.2 with KOH, 290 mOsmol). NMDA receptor-mediated EPSCs were recorded in Mg2+-free ACSF and neurons were held at −70 mV in the presence of CNQX (20 µM) and glycine (1 µM). The stimulation was delivered by a bipolar tungsten-stimulating electrode placed in layers V/VI of the ACC. Picrotoxin (100 µM) was always present to block GABAa receptor-mediated inhibitory synaptic currents in all experiments. Access resistance was 15–30 MΩ and monitored throughout the experiment. Data were discarded if access resistance changed 15% during an experiment. Data were filtered at 1 kHz and digitized at 10 kHz using the digidata 1440A. Data were collected and analyzed with Clampex and Clampfit 10.2 software (Axon Instruments).
Axon 200b amplifier
Manufactured by Molecular Devices
Sourced in United States
The Axon 200B amplifier is a laboratory instrument designed to measure and record electrical signals. It is capable of amplifying and filtering various types of electrophysiological signals, including those from patch-clamp recordings, voltage-sensitive dye measurements, and extracellular recordings.
Automatically generated - may contain errors
Lab products found in correlation
Pclamp 9, by Molecular Devices (4 mentions)
Clampfit 10, by Molecular Devices (3 mentions)
Bx51w1 microscope, by Olympus (1 mentions)
Clampex, by Molecular Devices (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Lsm 510 confocal system, by Zeiss (1 mentions)
Digidata 1322a, by Molecular Devices (1 mentions)
Bicuculline, by Bio-Techne (1 mentions)
Sylgard 184 silicone elastomer kit, by Dow (1 mentions)
Pclamp8, by Molecular Devices (1 mentions)
Fluo 4, by Thermo Fisher Scientific (1 mentions)
Labview, by National Instruments (1 mentions)
Clampfit version 10, by Molecular Devices (1 mentions)
Clampex software, by Molecular Devices (1 mentions)
Axon digidata 1440a, by Molecular Devices (1 mentions)
Clampex 10, by Molecular Devices (1 mentions)
Nir apo, by Nikon (1 mentions)
Pclamp software, by Molecular Devices (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Pc 10 pipette puller, by Narishige (1 mentions)
24 protocols using axon 200b amplifier
1
Whole-Cell Patch-Clamp Recording of Excitatory Postsynaptic Currents
2
TRPA1 Activation in HEK293 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (supplemented with 10% fetal bovine serum, penicillin, streptomycin and L-glutamine and transfected with expression vector using Lipofectamine™ LTX (Life Technologies). The cells were transfected with rat TRPA1 and 0.1 µg pGreen Lantern-1, and incubated with Dulbecco’s modified Eagle’s medium (supplemented with 10% fetal bovine serum, penicillin, streptomycin and L-glutamine) for 14 to 24 hours. The bath solutions for the patch-clamp experiments contained 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM glucose, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES), pH 7.4 (with NaOH). Calcium-free bath solutions for patch-clamp experiments contained 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 10 mM glucose, 5 mM ethylene glycol tetraacetic acid (EGTA), 10 mM HEPES, pH 7.4 (with NaOH). The pipette solution contained 140 mM KCl, 5 mM EGTA, and 10 mM HEPES, pH7.4 (with KOH). Data from whole-cell voltage-clamp recordings were sampled at 10 kHz and filtered at 5 kHz for analysis (Axon 200B amplifier with pCLAMP software, Axon Instruments). The cell was voltage-clamped at -60 mV. The current-voltage relationship was obtained using 500 ms voltage-ramp pulses from -100 to + 100 mV, applied every 5 sec. All experiments were performed at room temperature, except for eudesmol solution preparation described above.
3
Measuring Cardiomyocyte Action Potentials in Co-cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We measured AP properties of CMs in different co-cultures. Cells were constantly perfused with normal Tyrode’ solution at 37 °C (in mmol/L: NaCl 137, KCl 5.4, MgCl2 0.5, CaCl2 1.8, Na-HEPES 11.8, and glucose 10; pH 7.4) and patch pipettes (2–3 MΩ) (GB 200-8 P, Science Products) were filled with (in mmol/L): K-aspartate 120, NaCl 10, KCl 20, K-HEPES 10, MgATP 5, and K5Fluo-4 0.05; pH 7.2. Cell capacitance transients were recorded in the voltage-clamp mode (Axon 200B amplifier, Axon Instruments) during a 10 mV hyperpolarizing pulse (−70 mV to −80 mV for 150 ms) before and after heptanol (2 mM) application; cell capacitance was quantified by integrating the area under the curve of the transients. APs were recorded in the current-clamp mode during steady-state stimulation at 1 Hz (2 ms current injections). AP duration (APD) was quantified at 90% repolarization (APD90). Transfer of Fluo-4 from the CM to Fb/MyoFb was assessed in confocal imaging using a Zeiss LSM 510 confocal system. Transfer of Fluo-4 was assessed using a 40x/1.3 oil immersion objective during 488 nm excitation using an Argon laser. Fluo-4 images were combined with light microscopic images to contour cell edges.
4
Measuring Voltage-Gated Ion Channel Properties
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Currents were measured in the inside-out configuration using an Axon 200B amplifier and pClamp 9.0 (Axon Instruments, Inc.). Patches were recorded with 1–2 MΩ pipettes containing, in mM: 89 KCl, 15 HEPES, 0.4 CaCl2, and 0.8 MgCl2 (pH = 7.4 adjusted with KOH). The intracellular solution contained, in mM: 100 KCl, 10 HEPES, 1 EGTA, and 0.1 CaCl2, and, in µM: 0 or 100 cAMP (pH 7.1 adjusted with KOH). The internal solution was continuously perfused to the patches using a rapid perfusion system (Biologic Science Instruments RSC-160). From a holding potential of −80 mV, steps from +100 mV to −160 mV (in −20 mV steps) were applied to activate the current of the channel followed by a tail voltage of +40 mV to obtain the tail current. We measured the GV relation of channels by measuring the tail currents at +40 mV.
5
Electrophysiological Recording of GABA-Activated RPCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Glass coverslips plated with RPCs were transferred to a chamber containing the external solution (in mM: 125.0 NaCl, 2.5 KCl, 1.3 KH2PO4, 1.3 MgSO4, 25.0 NaHCO3, 2 CaCl2, 1.3 sodium L-ascorbate, 0.6 sodium pyruvate, 10 dextrose, pH 7.4, 320 mOsm), bubbled with 95% O2/5% CO2. Electrophysiological recordings were obtained at room temperature. RPCs were visualized by DIC microscopy. Microelectrodes (4–6 MΩ) were pulled from borosilicate glass capillaries and filled with the internal solution containing (in mM): 135 CsCl gluconate, 15 KCl, 4 MgCl2, 0.1 EGTA, 10.0 HEPES, 4 ATP magnesium salt, 0.3 GTP sodium salt, 7 phosphocreatine, pH 7.4, 300 mOsm. Data was collected using an Axon 200B amplifier and acquired with a DigiData 1322A (Axon Instruments) at 10 kHz. For measuring GABA-induced responses from RPCs, focal pressure ejection of 200 mM GABA or muscimol through a puffer pipette controlled by a Picospritze (2 s puff at 3–5 psi) was used to activate GABAaRs under the whole-cell voltage-clamp. All drugs were purchased from Sigma except bicuculline (100 μM; Tocris, UK).
6
Patch-Clamp Recordings of TRPV1 and TRPM8
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-cell patch-clamp recordings were performed 1 day after transfection. The standard bath solution was the same as that used in the Ca2+-imaging experiments; extracellular Ca2+ was removed and 5 mM EGTA was added for experiments in which our aim was to determine the dose-dependent effects of menthol or capsaicin. The pipetted solution contained 140 mM KCl, 5 mM EGTA, and 10 mM HEPES, pH 7.4 (adjusted with KOH). Data from the whole-cell voltage-clamp recordings were sampled at 10 kHz and filtered at 5 kHz for analysis (Axon 200B amplifier with pCLAMP software; Axon Instruments, Sunnyvale, CA). The membrane potential was clamped at −60 mV for all conditions. Voltage ramp-pulses from −150 to +100 mV (500 ms) were applied every 5 s for the inhibition of TRPV1 or TRPM8 activated by capsaicin or menthol, respectively, and every 2.5 s for the inhibition of TRPV1 or TRPM8 activated by temperatures or VBE, respectively.
7
Whole-Cell Voltage-Gated Currents in Pollen Tubes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pipettes were pulled from borosilicate glass blanks and coated with Sylgard (184 Silicone Elastomer kit; Dow Corning). Pipettes with solution had resistance values ranging from 15 to 35 MΩ in 10 mM CaCl2. Whole-cell currents across the plasma membrane of protoplasts isolated from pollen tubes were measured with an Axon 200B amplifier (Axon Instruments). Whole-cell preparations were obtained by forming a giga seal in the cell-attached mode, and the membrane was ruptured with a short burst of extra suction. A substantial increase in capacitance indicated that the whole-cell configuration was achieved, and the series resistance and capacitance were adjusted accordingly. Voltage-pulses of 2.5 s were used to elicit voltage-dependent currents. Data were sampled at 2 kHz and filtered at 0.5 kHz. Records were stored and analyzed by using pClamp 9.0 (Axon Instruments). The junction potential was corrected according to a published report (Amtmann and Sanders, 1997 (link)). All experiments were conducted at room temperature (20–22°C). The current-voltage curves were constructed by using the current values measured at the final voltage.
8
Non-Stationary Noise Analysis of Ion Channels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Currents in response to 100 sweeps to −120 (or −140) mV were recorded in cell-attached mode using an Axon 200B amplifier and pClamp8 (Axon Instruments Inc., San Jose, CA, USA). Micropatches were recorded with 0.5 MΩ pipettes containing (in mM) 100 KCl, 5 NaCl, 10 HEPES (pH = 7.4), 1 MgCl2, 1 CaCl2, 0.1 Lanthanum, and 0.1 Gadolinium. Non-stationary noise analyses [52 (link)] were calculated from the 100 sweeps using an in-house noise analysis program. Variances were calculated from the ensemble average of the squared differences in successive sweeps, δξ(t), to minimize errors due to drift or instability of the patch [53 (link),54 ]. Traces with excess noise due to, for example, large, endogenous single-channel currents were excluded if |δξ(t)| > 7[γ(V − Vrev)I + variancebackground]1/2, where γ is the estimated single-channel conductance [54 ]. Variance-to-mean curves were plotted and fitted with the standard parabolic equation var(I) = Ni2p(1 − p) = I(i − I/N) [52 (link)]. Control variance and mean currents were measured similarly in the presence of 100 µM of the HCN-channel-specific blocker ZD7288 and subtracted from the variance-to-mean data recorded in the absence of ZD7288 from the same patch. Minimum and maximum open probabilities were obtained from the fit of the variance to mean data from each patch and averaged.
9
Electrophysiological Recordings of Pyramidal Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Each slice was viewed under a microscope (Axioskop Fsmot; Zeiss, Jena, Germany) equipped with infrared differential interference contrast optics. Pyramidal cells in the bilateral PL/IL cortices were identified through a 40× water-immersion lens.
Voltage and current signals were recorded from EYFP-expressing pyramidal cells using an Axon 200B amplifier (Axon Instruments, Union City, CA). Action potentials (APs) and inhibitory post-synaptic potentials (IPSPs) were recorded in the current-clamp mode. The holding voltage for the excitatory post-synaptic currents (EPSCs) and APs was the same (–70 mV), and 0 mV for recording IPSCs. The aCSF contained the following (in mmol/L): 124 NaCl, 26 NaHCO3, 3.0 KCl, 1.0 NaH2PO4, 1.3 MgCl2, 1.5 CaCl2, 20 D-glucose, saturated with 95% O2 and 5% CO2. The pipette solution contained (in mmol/L) 120 potassium gluconate, 10 KCl, 4 ATP-Mg, 0.3 GTP, 10 HEPES, and 0.5 EGTA (pH 7.2, 270–280 mOsm with sucrose).
Voltage and current signals were recorded from EYFP-expressing pyramidal cells using an Axon 200B amplifier (Axon Instruments, Union City, CA). Action potentials (APs) and inhibitory post-synaptic potentials (IPSPs) were recorded in the current-clamp mode. The holding voltage for the excitatory post-synaptic currents (EPSCs) and APs was the same (–70 mV), and 0 mV for recording IPSCs. The aCSF contained the following (in mmol/L): 124 NaCl, 26 NaHCO3, 3.0 KCl, 1.0 NaH2PO4, 1.3 MgCl2, 1.5 CaCl2, 20 D-glucose, saturated with 95% O2 and 5% CO2. The pipette solution contained (in mmol/L) 120 potassium gluconate, 10 KCl, 4 ATP-Mg, 0.3 GTP, 10 HEPES, and 0.5 EGTA (pH 7.2, 270–280 mOsm with sucrose).
10
Patch-Clamp Recordings of Inner and Outer Hair Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-cell patch-clamp was achieved on IHCs or first- and second-row OHCs from middle to apical cochlea turns using an Axon 200B amplifier (Molecular Devices, Eugene, OR) with thick-walled borosilicate patch pipettes (2–6 MΩ) filled with an intracellular solution containing 125 mM CsCl, 3.5 mM MgCl2, 5 mM ATP, 5 mM creatine phosphate, 10 mM HEPES, 1 mM cesium BAPTA, and 3 mM ascorbate, pH = 7.2, 280–290 mOsm. For Ca2+ imaging, 0.5 mM Fluo-4 or Fluo-4FF (Invitrogen, Carlsbad, CA) and 0.08 mM Alexa 594 hydrazide (Invitrogen) was added to an internal mixture containing 85 mM CsCl, 3 mM MgCl2, 3 mM ATP, 5 mM creatine phosphate, 10 mM HEPES, 40 mM ascorbate, and 1 mM EGTA, pH = 7.2, 280–290 mOsm. Experiments were performed at 18–22°C. Whole cell currents were filtered at 10 kHz and sampled at 0.05–1 MHz using a model No. USB-6356 (National Instruments, Austin, TX) or a Personal DAQ3000 (IOtech, National Instruments) controlled by the software jClamp (SciSoft, http://www.scisoftco.com/jclamp.html ). Cells were voltage-clamped at −80 mV, not accounting for liquid junction potentials.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!