title: Isolating nuclei from cultured cells for patch-clamp electrophysiology of intracellular Ca(2+) channels.
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 × 10(6) 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.
authors.affiliation: Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Don-On Daniel
authors.initials: DO
authors.lastname: Mak
authors.forename: Horia
authors.initials: H
authors.lastname: Vais
authors.forename: King-Ho
authors.initials: KH
authors.lastname: Cheung
authors.forename: J Kevin
authors.initials: JK
authors.lastname: Foskett
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology*; D002413:Cations, Divalent / Q000378:metabolism; D018929:Cell Culture Techniques; D002460:Cell Line; D002951:Citrates / Q000378:metabolism; D003584:Cytological Techniques / Q000379:methods*; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism; D009685:Nuclear Envelope / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000379:methods*; D000077559:Sodium Citrate
chemical_list: D015220:Calcium Channels; D002413:Cations, Divalent; D002951:Citrates; D053496:Inositol 1,4,5-Trisphosphate Receptors; D000077559:Sodium Citrate; D002118:Calcium