Measuring Calcium Efflux in Isolated Primary Mitochondria

Mitochondrial calcium (mCa²⁺) homeostasis promotes oxidative metabolism within the physiological range; however, dysregulation can trigger necrotic cell death in diseases such as cardiac ischemia-reperfusion injury, muscular dystrophy, and neurodegenerative disorders, including Alzheimer's disease. It is widely understood that mitochondria exhibit rapid Ca²⁺ uptake primarily mediated by the mitochondrial calcium uniporter (MCU) complex, and that Ca²⁺ is exported via a combination of Na⁺/Ca²⁺ and H⁺/Ca²⁺ exchange processes. However, the proteins which mediate mCa²⁺ transport have only been partially identified. A particular challenge in determining which proteins mediate mCa²⁺ efflux and their relative contributions to mCa²⁺ homeostasis is the lack of a clear, reproducible assay for mCa²⁺ efflux applicable across genotypes. Here, we provide instructions for an optimized fluorometric method to measure mCa²⁺ efflux in isolated mitochondria that establishes robust Ca²⁺ efflux signals, differentiates between total and Na⁺-independent Ca²⁺ efflux modalities, and generates highly reproducible data, allowing comparisons across tissues and genotypes. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Total Calcium Efflux Assay in Isolated Mitochondria Alternate Protocol 1: Na⁺-Independent Calcium Efflux Assay in Isolated Mitochondria Support Protocol 1: Isolation of Cardiac Mitochondria Support Protocol 2: Isolation of Skeletal Muscle Mitochondria Support Protocol 3: Isolation of Liver Mitochondria Support Protocol 4: Isolation of Cell Line Mitochondria Basic Protocol 2: Analysis of Calcium Efflux.