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

Abstract Optimum cytosolic calcium concentrations support balanced mitochondrial respiration. However, cytosolic Ca 2+ concentrations vary among cell types and excess Ca 2+ can cause mitochondrial dysfunction. We optimized an isolation protocol to eliminate excess Ca 2+ and thereby minimizing structural damage. Ca 2+ uptake was monitored by measuring mitochondrial Ca 2+ -dependent PKA activity using cAMP ELISAs, and O 2 consumption levels during mitochondrial respiration using high-resolution respirometry. 3 nM Ca 2+ 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 Ca 2+ -dependent regulation of mitochondrial signaling.