Isolating Nuclei from Cultured Cells for Patch-Clamp Electrophysiology of Intracellular Ca2+ Channels
- 1Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104;
- 2Department of Physiology, University of Hong Kong, Hong Kong
Abstract
Nuclear patch-clamp experiments can be performed with intact nuclei or with nuclei from which the outer nuclear membrane has been removed. This protocol presents procedures for harvesting different types of cultured cells, isolating nuclei, and exposing the inner nuclear membrane by agitating in the presence of sodium citrate. Particulars about obtaining and maintaining the cells of interest in culture are not described here. However, care should be taken not to allow the cells to grow beyond a density of 2–3 × 106 cells/mL because this may decrease both the cell viability and the success rate of detecting active inositol 1,4,5-trisphosphate receptor (InsP3R) channels in nuclear patches.
Footnotes
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↵3 Correspondence: foskett{at}mail.med.upenn.edu
- © 2013 Cold Spring Harbor Laboratory Press
