Mt-fura-2, a Ratiometric Mitochondria-Targeted Ca2+ Sensor. Determination of Spectroscopic Properties and Ca2+ Imaging Assays

Ca2+ handling by mitochondria is implicated in energy production, shaping of cytosolic Ca2+ rises, and determination of cell fate. It is therefore of crucial interest for researchers to directly measure mitochondrial Ca2+ concentration [Ca2+] in living cells. Synthetic fluorescent Ca2+ indicators, providing a straightforward loading technique, represents a tempting strategy. Recently, we developed a new highly selective mitochondria-targeted Ca2+ indicator named mt-fura-2, obtained by coupling two triphenylphosphonium cation-containing groups to the molecular backbone of the cytosolic ratiometric Ca2+ indicator fura-2. The protocols we describe here cover all the significant steps that are necessary to characterize the probe and apply it to biologically relevant contexts. The procedures reported are referred to mt-fura-2 but could in principle be applied to characterize other mitochondria-targeted Ca2+ probes. More in general, with the due modifications, this chapter can be considered as a handbook for the characterization and/or application of mitochondria-targeted chemical Ca2+ probes.