Abstract
During a conventional whole-cell patch clamp experiment, diffusible cytosolic ions or molecules absent in the pipette solution can become diluted by a factor of one million or more, leading to diminished current or fluorescent signals. Existing methods to prevent or limit cytosol diffusion include reducing the diameter of the pipette's orifice, adding cytosolic extract or physiological entities to the pipette solution, and using the perforated patch clamp configuration. The first method introduces measurement error in recorded signals from increased series resistance and the latter two are cumbersome to perform. In addition, most perforated patch configurations, prevent investigators from using test compounds in the pipette solution. We present a method to overcome these limitations by minimizing cytosol dilution using a novel pipette holder. Cell-attached configuration is obtained with the pipette filled with pipette solution. Most of the pipette solution is then replaced with mineral oil so that cytosol dilution can be minimized in whole-cell configuration. To accomplish this requires a suction line and two Ag/AgCl electrodes inside the pipette. Testing our novel pipette holder with Chinese Hamster Ovarian cells, we demonstrate cytosol dilution factors between 76 and 234. For large cells with somas greater than 40μm, cytosol dilution factors of 10 or less are achievable.
Highlights
The whole-cell patch-clamp configuration is a variant of the patch-clamp technique [1] [2], used to study membrane embedded proteins and their role in cellular physiology and Manuscript received September 16, 2012
The Ag/AgCl wire connects to electronic circuitry, which either clamps a cell’s membrane voltage with respect to a ground Ag/AgCl electrode placed in the extracellular medium or clamps the current passing through the membrane
In order to achieve a low resistance whole-cell configuration while minimizing cytosol dilution, we reduce the volume of the pipette solution by using a mechanohydraulic method implemented with a novel pipette holder
Summary
The whole-cell patch-clamp configuration is a variant of the patch-clamp technique [1] [2], used to study membrane embedded proteins and their role in cellular physiology and Manuscript received September 16, 2012. In this configuration, the physical system is a whole-cell surrounded by an extracellular medium and an essential part of the probing instrument is a glass micropipette containing an electrolyte solution and an electrochemical transducer, an Ag/AgCl wire (Fig. 1). The Ag/AgCl wire connects to electronic circuitry, which either clamps a cell’s membrane voltage with respect to a ground Ag/AgCl electrode placed in the extracellular medium or clamps the current passing through the membrane. A length of ~7 mm of mineral oil is filled in the pipette above the interface between oil and solution.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
