Practical aspects of measuring intracellular calcium signals with fluorescent indicators

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.