High-throughput functional assays of IP3-evoked Ca2+ release
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Updated: May 7, 2026
Imaging Local Ca2+ Signals in Cultured Mammalian Cells
Published on: March 3, 2015
Stephen C Tovey1, Colin W Taylor
1Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, United Kingdom.
Abstract:
This protocol describes procedures for high-throughput functional analyses of inositol 1,4,5-trisphosphate receptors (IP3Rs) in permeabilized cells. The methods are applicable to native IP3Rs in a variety of cells and to recombinant IP3Rs stably expressed in DT40 cells in which gene disruption has abolished expression of endogenous IP3Rs. A low-affinity Ca(2+)-indicator (Mag-Fluo-4) trapped within the endoplasmic reticulum (ER) of permeabilized cells is used to report changes in luminal free Ca(2+) concentration. A fluorescence plate-reader equipped to allow automated additions permits rapid measurements of the Ca(2+) release evoked by IP3R. The procedure can be completed in 2-3 h.
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