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

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.