Analyzing Mitochondrial Calcium Influx in Isolated Mitochondria

Mitochondria play a crucial role in Ca²⁺ signaling and homeostasis and can contribute to shaping the cytosolic Ca²⁺ landscape as well as regulate a variety of pathways including energy production and cell death. Dysregulation of mitochondrial Ca²⁺ homeostasis promotes pathologies including neurodegenerative diseases, cardiovascular disorders, and metabolic syndromes. The significance of mitochondria to Ca²⁺ signaling and regulation underscores the value of methods to assess mitochondrial Ca²⁺ import. Here we present a plate reader-based method using the Ca²⁺-sensitive fluorescent probe calcium green-5 N to measure mitochondrial Ca²⁺ import in isolated cardiac mitochondria. This technique can be expanded to measure Ca²⁺ uptake in mitochondria isolated from other tissue types and from cultured cells.