JoVE Encyclopedia of Experiments
Biological Techniques
0 views • 3:13 min • July 8th, 2025
Mitochondria take up cytosolic calcium as it is essential for their proper functioning.
Excessive calcium influx into the mitochondrial matrix - mitochondrial calcium overload - triggers the opening of an inner membrane channel, the mitochondrial permeability transition pore, or MPTP. The opened pore allows increased calcium efflux from the mitochondria, causing mitochondrial dysfunction.
To measure the mitochondrial calcium uptake, begin with a multi-well plate containing isolated mitochondria suspended in a suitable buffer. Supplement the well with pyruvate and malate, which are ATP-producing substrates. Mix the contents of the well, and incubate.
During incubation, pyruvate and malate are converted to ATPs, energizing the mitochondria. Add a solution containing mitochondrial membrane-impermeable, calcium-sensitive fluorescent dye molecules. Dispense the calcium-containing buffer to the energized mitochondria.
Monitor the dye's calcium fluorescence as the dye molecules bind to the free calcium in the buffer.
Add more calcium ions to initiate mitochondrial calcium influx. The gradually increasing mitochondrial calcium decreases the calcium ions in the buffer, reducing the dye's ca
View the full transcript and gain access to thousands of scientific videos