使用荧光指示剂测量细胞内钙信号的实际操作

Practical aspects of measuring intracellular calcium signals with fluorescent indicators.

作者信息

Kao Joseph P Y, Li Gong, Auston Darryl A

机构信息

Center for Biomedical Engineering and Technology, and Department of Physiology University of Maryland School of Medicine, Baltimore, Maryland, USA.

出版信息

Methods Cell Biol. 2010;99:113-52. doi: 10.1016/B978-0-12-374841-6.00005-0.

DOI:10.1016/B978-0-12-374841-6.00005-0

PMID:21035685

Abstract

The use of fluorescent indicators for monitoring calcium (Ca(2+)) signals and for measuring Ca(2+) concentration ([Ca(2+)]) in living cells is described. The following topics are covered in detail: (1) ratiometric and nonratiometric fluorescent indicators and the principles underlying their use, (2) techniques for loading Ca(2+) indicators and Ca(2+) buffers into living cells, (3) calibration of indicator fluorescence intensity measurements to yield values of intracellular [Ca(2+)], (4) analysis of nonratiometric fluorescence intensity data and caveats relating to their interpretation, (5) techniques for manipulating intracellular and extracellular [Ca(2+)], and (6) the use of fluorescent indicators to monitor Ca(2+) signals in mitochondria. The chapter aims to present these fundamental topics in a manner that is practically useful and intuitively accessible. The origins of key mathematical equations used in the article are outlined in two appendices.

摘要

本文描述了使用荧光指示剂监测活细胞中的钙（Ca(2+)）信号以及测量Ca(2+)浓度（[Ca(2+)]）的方法。详细涵盖了以下主题：（1）比率型和非比率型荧光指示剂及其使用的基本原理，（2）将Ca(2+)指示剂和Ca(2+)缓冲剂加载到活细胞中的技术，（3）校准指示剂荧光强度测量以获得细胞内[Ca(2+)]值，（4）非比率型荧光强度数据分析及其解释的注意事项，（5）操纵细胞内和细胞外[Ca(2+)]的技术，以及（6）使用荧光指示剂监测线粒体中的Ca(2+)信号。本章旨在以实际有用且直观易懂的方式呈现这些基本主题。文章中使用的关键数学方程的起源在两个附录中进行了概述。