Tovey Stephen C, Taylor Colin W
Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, United Kingdom.
Cold Spring Harb Protoc. 2013 Oct 1;2013(10):930-7. doi: 10.1101/pdb.prot073072.
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.
本方案描述了通透细胞中肌醇1,4,5-三磷酸受体（IP3Rs）高通量功能分析的程序。这些方法适用于多种细胞中的天然IP3Rs，以及在DT40细胞中稳定表达的重组IP3Rs，其中基因破坏已消除内源性IP3Rs的表达。被困在通透细胞内质网（ER）中的低亲和力钙指示剂（Mag-Fluo-4）用于报告腔内游离钙浓度的变化。配备自动添加功能的荧光酶标仪可快速测量IP3R诱发的钙释放。该程序可在2-3小时内完成。
