Minimizing Cytosol Dilution in Whole-Cell Patch-Clamp Experiments
Samsoon Inayat, Lawrence H. Pinto, and John B. Troy
Volume: 60, Issue: 7, Page(s): 2042-2051
The cell is the basic unit of life. While living, it responds to diverse internal and external stimuli and exchanges materials across its plasma membrane through various types of pores and processes. In multicellular organisms such as humans, numerous kinds of diseases occur due to malfunction of these pores and processes. In order to study them and develop therapeutic drugs, the whole-cell patch-clamp technique is widely used. This technique allows the identification of the types of ions and the measurement of their flow rates across cell membranes of cells ranging in size from a few to tens of micrometers. The probing instrument consists of a glass pipette with a tip opening of about 1-2 micrometers (1/100th the diameter of a human hair). The pipette, filled with a solution, has its tip pressed against the cell membrane. Suction then applied inside the pipette gives cell-attached configuration, where the cell membrane seals and gets entrapped in the pipette tip. Further suction applied inside the pipette gives whole-cell configuration, where the cell membrane entrapped in the tip breaks and the pipette solution forms a continuum with the intracellular solution, the cytosol. As the volume of the pipette solution is usually about one million or more times that of the cytosol, diffusible cytosolic ions or molecules absent in the pipette solution become diluted leading to errors in the observed signals. Existing remedies to avoid dilution, briefly discussed in the paper, introduce other measurement errors. We introduce a device with which we minimize the pipette solution before breaking the cell membrane entrapped in the pipette tip in order to minimize cytosol dilution. We demonstrate pipette solution volumes ranging between 76 and 234 times that of the cytosol as a technical advance compared to the conventional method.