An improved method for isolation of mitochondria from cell lines that enables reconstitution of calcium-dependent processes.

Optimum cytosolic calcium concentrations support balanced mitochondrial respiration. However, cytosolic Ca2+ concentrations vary among cell types and excess Ca2+ can cause mitochondrial dysfunction. We optimized an isolation protocol to eliminate excess Ca2+ and thereby minimizing structural damage. Ca2+ uptake was monitored by measuring mitochondrial Ca2+-dependent PKA activity using cAMP ELISAs, and O2 consumption levels during mitochondrial respiration using high-resolution respirometry. 3 nM Ca2+ was found to increase cAMP levels and produce optimal state III respiration. Hence, optimized isolation of mitochondria from cell lines using calcium denudation provides the best platform for the study of Ca2+-dependent regulation of mitochondrial signaling.