The overall goal of this procedure is to measure mitochondrial calcium influx in isolated mitochondria and cultured cells. This method can help answer key questions in the calcium signaling field such as how mitochondria help to shape the cytosolic calcium landscape, and the role of mitochondrial calcium dynamics in disease conditions. The main advantage of this technique is that our protocols can be adapted to a variety of model systems to assess mitochondrial calcium influx capacity.

I will be demonstrating the plate-reader-based mitochondrial calcium uptake procedure. And Dr.Joshua Maxwell, an instructor in the Department of Pediatrics will be demonstrating the confocal-microscope-based procedure to measure mitochondrial calcium influx. Start this experiment by thoroughly rinsing previously isolated heart in 25 milliliters of ice-cold one-X PBS making sure that the blood is completely squeezed out of the ventricles.

In five milliliters of ice-cold one-X PBS, using a pair of sharp scissors, mince the heart tissue into small pieces. Carefully discard the PBS and transfer the tissue to a pre-chilled seven-milliliter glass Teflon Dounce homogenizer. Then, add five milliliters of ice-cold MSEGTA buffer and homogenize until there are no visible tissue pieces.

Transfer the homogenate to a 15-milliliter tube, and then centrifuge at 600 times G at four degrees Celsius for five minutes to pellet unwanted nuclei and unbroken cells. Transfer the supernatant to a fresh 15-milliliter tube, and centrifuge at 10, 000 times G at four degrees Celsius for 10 minutes. After discarding the supernatant, keep the mitochondrial pellet on ice.

To wash the pellet, add five milliliters of ice-cold MSEGTA buffer to it, resuspend and then centrifuge at 10, 000 times G at four degrees Celsius for 10 minutes. After discarding the supernatant, repeat the wash and keep the pellet on ice. To start mitochondrial calcium uptake measurement using a plate reader, program the reader to perform a kinetic read of Calcium Green-5N florescence every second for a total time of 1, 000 seconds.

Program the reagent injectors to dispense five 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 one microliter of one molar pyruvate, one microliter of 500-millimolar malate and one microliter of one-millimolar Calcium Green-5N stock and mix gently by pipetting. Incubate protected from light at room temperature for two minutes for mitochondria to become energized. Finally, start the pre-programmed kinetic protocol and monitor Calcium Green-5N florescence.

For this part of the protocol, prepare Rhod-2 AM MitoTracker Green working solution by mixing 20 microliters of Rhod-2 AM, 0.2 microliters of MitoTracker Green, 2.5 microliters of 20%pluronic F-127 and one milliliter of Tyrode solution. Onto a previously-prepared coverslip, plated with NIH/3T3 cells, add the Rhod-2 AM MitoTracker Green solution drop-wise until covered. For the cells to load with the dyes, incubate the coverslip protected from light for 30 minutes at room temperature.

To deesterify Rhod-2 AM, gently remove the Rhod-2 AM MitoTracker Green solution, and replace it with approximately 300 microliters of fresh Tyrode solution to cover the cells. Incubate the coverslip protected from light for 30 minutes at room temperature. Now, transfer the coverslip to the microscope imaging chamber and fill the chamber with wash solution.

Adjust the focus to observe the cells and phase contrast at 40X magnification. To permeabilize the plasma membrane of Rhod-2 AM MitoTracker Green loaded cells, remove the wash solution from the coverslip and replace it with approximately 300 microliters of permeabilization solution to cover the cells. Monitor the plasma membrane morphology during this process.

When permeabilized, cells will develop a roughened surface. Remove the permeabilization solution immediately after complete permeabilization and replace it with zero-calcium internal solution. Next, focus on permeabilized cells displaying a clear co-localization of Rhod-2 and MitoTracker Green to image Rhod-2 and Mitotracker Green florescence simultaneously.

Next, decrease the microscope laser and gain settings to make the mitochondrial Rhod-2 florescence dim and barely visible. To capture the kinetics of mitochondrial calcium changes, select microscope settings to acquire two-dimensional scans at an appropriate frame-rate and time course. Remove the zero-calcium internal solution making sure not to disturb the cells and microscope focus and then start image acquisition.

Using a syringe, manually add calcium-replete internal solution at the 10-seconds time point. Finally, in the image acquisition software, select regions of interest to encompass regions of co-localization of Rhod-2 and MitoTracker Green signal. When calcium chloride is added to isolated cardiac mitochondria, florescence from the calcium dye increases as mitochondria uptake calcium from the buffer florescence decreases.

However, at the third edition of calcium, florescence suddenly starts increasing after a drop indicating mitochondrial calcium overload and mitochondrial permeability transition pore opening which can lead to mitochondrial inner membrane permeabilization. On the other hand, when mitochondria are pre-treated with an inhibitor of mitochondrial calcium uptake, there is an increase in florescence following each calcium addition. In cultured NIH/3T3 cells, the mitochondrial network is stained with MitoTracker Green dye.

Cells are also stained with Rhod-2 dye and after saponin permeabilization, cytosolic Rhod-2 is washed away leaving just mitochondrial staining which co-localizes with the MitoTracker. Since Rhod-2 can accumulate in non-mitochondrial structures, the analysis should focus only on regions where MitoTracker and Rhod-2 co-localize. The addition of calcium causes an increase in Rhod-2 florescence in control cells whereas in cells treated with the inhibitor of mitochondrial calcium uptake, there is no change in florescence.

These protocols can be applied to measure mitochondrial calcium uptake in many cell types including primary cells such as cardiac myocytes and neurons in immortalized cell lines as well as isolated mitochondria from many tissue types including heart, liver and brain. Calcium handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in different cells. The ability to accurately measure the influx of calcium from mitochondria is an important part of determining the role of mitochondrial calcium handling in these processes.

After watching this video, you should have a good understanding of how to measure mitochondrial calcium influx in isolated mitochondria and cultured cells.