Whole-cell patch-clamp experiments on HEK cells were performed 16 to 24 hours after transfection with calcium phosphate (69). The internal pipette solution contained the following: 110 mM CsCl, 3 mM MgCl2, 10 mM EGTA, 10 mM Hepes, 5 mM MgATP, and 1 mM GTP (pH 7.2 adjusted with CsOH). The bath solution contained the following: 20 mM BaCl2, 1 mM MgCl2, 10 mM Hepes, 40 mM TEA-Cl, 65 mM CsCl, and 10 mM d-glucose (pH 7.4 adjusted with TEA-OH) for HEK cells and 10 mM BaCl2, 10 mM Hepes, 120 mM TEA-Cl, and 10 mM d-glucose (pH 7.4 adjusted with TEA-OH) for DRG. The DRG recording solution also contained 10 μM nifedipine (Sigma, USA) to inhibit L-type calcium currents. The recording solutions used for TRPV1 currents contained the following: 140 mM NaCl, 1.5 mM CaCl2, 2 mM MgCl2, 5 mM KCl, 10 mM Hepes, and 10 mM d-glucose (pH 7.4 adjusted with NaOH) and the same internal solution mentioned above. In cells that showed TRPV1 currents, the external solution was switched to the barium-containing solution mentioned above to record voltage-gated calcium currents. Patch-clamp experiments were performed using an Axopatch 200B amplifier (Axon Instruments), and pClamp 10.5 software was used for data acquisition and analysis (both from Molecular Devices Corp.). Data were digitized at 10 kHz and low-pass–filtered at 1 kHz. Borosilicate glass (Harvard Apparatus Ltd.) pipettes were pulled and polished to a resistance of 2 to 5 megohms with a DMZ-Universal Puller (Zeitz-Instruments GmbH.). When applicable, voltages were corrected for liquid junction potentials. All experiments were conducted at room temperature (22° ± 2°C).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.