Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in cytosolic Ca²⁺ buffering, and the ATP production in mitochondrial can be mediated by intramitochondrial free Ca²⁺ concentration. Ca²⁺ retention capacity can be regarded as the capability of mitochondria to retain calcium in the mitochondrial matrix. Accumulated intracellular Ca²⁺ leads to the permeability of the inner mitochondrial membrane, termed the opening of mitochondrial permeability transition pore (mPTP), which leads to the leakage of molecules with a molecular weight less than 1.5 kDa. Ca²⁺-triggered mitochondria swelling is used to indicate the mPTP opening. Here, we describe two assays to examine the Ca²⁺ retention capacity and Ca²⁺-triggered mitochondrial swelling in isolated mitochondria. After certain amounts of Ca²⁺ are added, all steps can be completed in one day and recorded by a microplate reader. Thus, these two simple and effective assays can be adopted to assess the Ca²⁺-related mitochondrial functions.