title: Minimizing cytosol dilution in whole-cell patch-clamp experiments.
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 40 μm, cytosol dilution factors of 10 or less are achievable.
authors: Biomedical Engineering Department, Northwestern University, Evanston, IL 60208, USA. sam@northwestern.edu
authors: Samsoon
authors: S
authors: Inayat
authors: Lawrence H
authors: LH
authors: Pinto
authors: John B
authors: JB
authors: Troy
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003600:Cytosol / Q000737:chemistry*; D004058:Diffusion; D004867:Equipment Design; D019544:Equipment Failure Analysis; D018408:Patch-Clamp Techniques / Q000295:instrumentation* / Q000379:methods*