Measuring Calcium Efflux in Isolated Primary Mitochondria

Abstract Mitochondrial calcium (mCa 2+ ) 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 Ca 2+ uptake primarily mediated by the mitochondrial calcium uniporter (MCU) complex, and that Ca 2+ is exported via a combination of Na + /Ca 2+ and H + /Ca 2+ exchange processes. However, the proteins which mediate mCa 2+ transport have only been partially identified. A particular challenge in determining which proteins mediate mCa 2+ efflux and their relative contributions to mCa 2+ homeostasis is the lack of a clear, reproducible assay for mCa 2+ efflux applicable across genotypes. Here, we provide instructions for an optimized fluorometric method to measure mCa 2+ efflux in isolated mitochondria that establishes robust Ca 2+ efflux signals, differentiates between total and Na + ‐independent Ca 2+ 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