Calcium Influx Assay to Measure Mitochondrial Calcium Uptake in Cultured Cells

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's 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 de-esterify Rhod-2/AM, gently remove the Rhod-2/AM-MitoTracker Green solution, and replace it with approximately 300 microliters of fresh Tyrode's 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 fluorescence simultaneously.

Next, decrease the microscope laser and gain settings to make the mitochondrial Rhod-2 fluorescence 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.