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The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in cytosolic Ca 2+ buffering, and the ATP production in mitochondrial can be mediated by intramitochondrial free Ca 2+ concentration. Ca 2+ retention capacity can be regarded as the capability of mitochondria to retain calcium in the mitochondrial matrix. Accumulated intracellular Ca 2+ 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. Ca 2+ -triggered mitochondria swelling is used to indicate the mPTP opening. Here, we describe two assays to examine the Ca 2+ retention capacity and Ca 2+ -triggered mitochondrial swelling in isolated mitochondria. After certain amounts of Ca 2+ 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 Ca 2+ -related mitochondrial functions.

Mitochondrial Ca<sup>2+</sup> Retention Capacity Assay and Ca<sup>2+</sup>-triggered Mitochondrial Swelling Assay