Measuring the in situ Kd of a genetically encoded Ca2+ sensor

The use of genetically encoded Ca(2+) sensors (GECIs) for long-term monitoring of intracellular Ca(2+) has become increasingly common in the last decade. Emission-ratiometric GECIs, such as those in the Yellow Cameleon family, can be used to make quantitative measurements, meaning that their fluorescence signals can be converted to free Ca(2+) concentrations ([Ca(2+)]free). This conversion is only as accurate as the sensor's apparent dissociation constant for Ca(2+) (K'd), which depends on temperature, pH, and salt concentration. This protocol describes a method for performing a titration, in living cells (in situ), of cytosolic, nuclear, or mitochondrial sensors.