Mitochondrial Calcium Uptake Assay: A Plate Reader-Based Method for Measuring Calcium Uptake in Isolated Mitochondria

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.

To start mitochondrial calcium uptake measurement using a plate reader, program the reader to perform a kinetic read of calcium green-5N fluorescence every second, for a total time of 1,000 seconds.

Program the reagent injectors to dispense 5 microliters of calcium chloride solution at various time points. Then, prime the injectors with the calcium chloride solution that will be used.

Next, add 200 micrograms of isolated mitochondria to one well of a 96-well plate, and add the appropriate volume of potassium chloride buffer to achieve a total volume of 197 microliters. Then, add 1 microliter of 1 molar pyruvate, 1 microliter of 500 millimolar malate, and 1 microliter of 1 millimolar calcium green-5N stock, and mix gently by pipetting.

Incubate protected from light at room temperature for 2 minutes, for mitochondria to become energized. Finally, start the pre-programmed kinetic protocol, and monitor calcium green-5N fluorescence.