Calcium Influx Assay to Measure Mitochondrial Calcium Uptake in Cultured Cells

The mitochondrial uptake of cytosolic calcium ions via its inner membrane mitochondrial uniporter complex is crucial for its functioning.

To measure the mitochondrial calcium influx, begin with a multi-well plate containing an ECM-coated coverslip at the bottom of its well. Plate the fibroblast suspension onto the coverslip, and allow the cells to adhere to the ECM.

Add a dye mix containing an inactive, esterified red-fluorescent calcium-sensitive dye and a green-fluorescent mitochondria-selective dye. Incubate to allow the dye molecules to enter the cell cytosol.

The mitochondria-sensitive dye selectively diffuses through its membrane and localizes in the mitochondrial matrix. The calcium-sensitive dye spreads throughout the mitochondria and extra-mitochondrial spaces. Inside the mitochondrial lumen, endogenous esterases cleave the ester moiety from the red dye, releasing a membrane-impermeable red fluorophore that remains trapped inside the mitochondria.

Add a surfactant-containing permeabilization solution. At low concentrations, surfactant molecules restrictively dissolve the cells' plasma membrane cholesterol, leaving their mitochondria intact and creating spaces through which calcium-sensitive dye molecules leak out of the cytoplasm.  This selectively retains the calcium-sensitive red fluorophores and mitochondria-specific green fluorophores in the mitochondria.

Transfer the coverslip to an imaging chamber fixed on a confocal microscope, and add a calcium-containing buffer. Within the permeabilized cells, locate regions displaying co-localized red and green fluorescence, indicating mitochondria-localized calcium. The red fluorescence gradually augments, depicting progressive mitochondrial calcium uptake.

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.