Ca²⁺ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca²⁺ from mitochondria is important for determining the role of mitochondrial Ca²⁺ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca²⁺ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca²⁺ uptake using the Ca²⁺ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca²⁺ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca²⁺ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca²⁺ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca²⁺ in permeabilized cells using the Ca²⁺ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca²⁺. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca²⁺ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca²⁺ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

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