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 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.