Measuring calcium signaling using genetically targetable fluorescent indicators

Genetically encoded Ca(2+) indicators allow researchers to quantitatively measure Ca(2+) dynamics in a variety of experimental systems. This protocol summarizes the indicators that are available, and highlights those that are most appropriate for a number of experimental conditions, such as measuring Ca(2+) in specific organelles and localizations in mammalian tissue-culture cells. The protocol itself focuses on the use of a cameleon, which is a fluorescence resonance-energy transfer (FRET)-based indicator comprising two fluorescent proteins and two Ca(2+)-responsive elements (a variant of calmodulin (CaM) and a CaM-binding peptide). This protocol details how to set up and conduct a Ca(2+)-imaging experiment, accomplish offline data processing (such as background correction) and convert the observed FRET ratio changes to Ca(2+) concentrations. Additionally, we highlight some of the challenges in observing organellar Ca(2+) and the alternative strategies researchers can employ for effectively calibrating the genetically encoded Ca(2+) indicators in these locations. Setting up and conducting an initial calibration of the microscope system is estimated to take similar to 1 week, assuming that all the component parts are readily available. Cell culture and transfection is estimated to take similar to 3 d (from the time of plating cells on imaging dishes). An experiment and calibration will probably take a few hours. Finally, the offline data workup can take similar to 1 d depending on the extent of analysis.