Mitochondrial Calcium Uptake Assay: A Plate Reader-Based Method for Measuring Calcium Uptake in Isolated Mitochondria
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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 calcium fluorescence.
Once the mitochondria reach their maximum calcium-carrying capacity, the further addition of calcium ions open the MPTP channels and import calcium into the buffer. The buffer calcium binds to the dye molecules, causing increased dye calcium fluorescence.
Plot the dye fluorescence at different time points of calcium addition to monitor the kinetics of mitochondrial calcium uptake.
