Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells.

Ca 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 Ca from mitochondria is important for determining the role of mitochondrial Ca handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca uptake using the Ca 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 Ca from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca 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 Ca in permeabilized cells using the Ca 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 Ca. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.