GlyT1, GlyT2, GlyRα1, GlyRα3, GlyRα1β and GlyRα3β are electrogenic, allowing activation to be measured via the two-electrode voltage clamp technique. Oocytes were voltage clamped at −60 mV, and whole-cell currents generated by the substrate were recorded with a Geneclamp 500 amplifier (Axon Instruments, Foster City, CA, USA), digitised by a Powerlab 2/20 chart recorder (ADInstruments, Sydney, Australia). LabChart version 8 software (ADInstruments, Sydney, Australia) was used to visualise and process current traces. Recordings were performed in frog Ringer’s solution, except for low Na+ experiments where Na+ was replaced with choline. Oocytes were placed in an oval-shaped bath with a volume of 0.3 mL, with laminar flow around the oocyte at a rate of 12 mL min−1 under gravity feed.
Powerlab 2 20 chart recorder
Manufactured by ADInstruments
Sourced in United States
The PowerLab 2/20 is a data acquisition system designed for recording and analyzing physiological signals. It features 8 isolated analog input channels capable of recording a wide range of signals, as well as digital inputs and outputs for integration with external devices. The PowerLab 2/20 is capable of sampling at rates up to 200 kHz and provides real-time data display and analysis capabilities.
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Lab products found in correlation
4 protocols using powerlab 2 20 chart recorder
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Electrogenic Neurotransmitter Receptor Assay
2
Xenopus Oocyte Glycine Transporter Assay
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Oocytes were extracted from female Xenopus laevis frogs and detached from follicle cell containing lobes by digestion with 2 mg/mL collagenase A (Boehringer, Mannheim, Germany). Defoliculated stage V-VI oocytes were injected with 4.6 ng of cRNA encoding WT or mutant transporter (Drummond Nanoinject, Drummond Scientific Co., Broomall, PA, USA). Surgical proceedures have been approved by the University of Sydney Animal Ethics Committee (protocol 2016/970). The oocytes were stored at 16–18°C for 2–5 days in ND96 solution (96 mM NaCl, 2 mM KCL, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES, pH 7.55), supplemented with 2.5 mM sodium pyruvate, 0.5 mM theophylline, 50 µg/mL gentamicin and 100 µM/mL tetracycline.
2–5 days following injection, glycine transport currents were measured at −60 mV using Geneclamp 500 amplifier (Axon Instruments, Foster City, CA, USA) with a Powerlab 2/20 chart recorder (ADInstruments, Sydney, Australia) and chart software (ADInstruments). All data were subsequently analysed using GraphPad Prism 7.02 (GraphPad Software, San Diego, CA).
2–5 days following injection, glycine transport currents were measured at −60 mV using Geneclamp 500 amplifier (Axon Instruments, Foster City, CA, USA) with a Powerlab 2/20 chart recorder (ADInstruments, Sydney, Australia) and chart software (ADInstruments). All data were subsequently analysed using GraphPad Prism 7.02 (GraphPad Software, San Diego, CA).
3
Two-Electrode Voltage Clamp Assay
4
Two-Electrode Voltage Clamp Assay
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2–4 days after injections, currents were recorded using the two-electrode voltage clamp technique with a Geneclamp 500 amplifier (Axon Instruments, Foster City, CA, USA) interfaced with a PowerLab 2/20 chart recorder (ADInstruments, Sydney, Australia) and a Digidata 1322A (Axon Instruments, CA, USA), which was used in conjunction with Chart software (ADInstuments; Axon instruments). All recordings were made with a bath grounded via a 3 M KCl/ 1% agar bridge linking to a 3 M KCl reservoir containing a Ag/AgCl2 ground electrode to minimize offset potentials. Current-voltage relationships for substrate-elicited conductance were determined by measuring substrate-elicited currents during 245 ms voltage pulses between −100 mV and +60 mV at 10 mV steps. Background currents were eliminated by subtracting currents in the absence of substrate from substrate-elicited currents at corresponding membrane potentials. For cross-linking studies, current elicited by an initial control dose of 100 μM L-glutamate was recorded prior to 2 min incubation with 1 mM DTT. Currents were recorded following 1 mM DTT treatment and following subsequent 5 min incubation with 400 μM CuPh. For hydrophobic gate mutagenesis studies, current elicited was measured in Ringer’s solution containing 30 μM aspartate.
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