Calcium Influx Assay to Measure Mitochondrial Calcium Uptake in Cultured Cells

1. Reagents and Solutions

NOTE: The second part is experimental procedure for confocal imaging of mitochondrial Ca²⁺ in cultured cells

1. Make Tyrode's solution (130 mM NaCl, 4 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose, and 10 mM HEPES, pH 7.2 with KOH). Store it at 4 °C. Warm it to room temperature before use.
2. Prepare Wash Solution (100 mM potassium acetate, 15 mM KCl, 5 mM KH₂PO₄, 5 mM Mg-ATP, 0.35 mM EGTA, 0.12 mM CaCl₂, 0.75 mM MgCl₂, 10 mM phosphocreatine, 10 mM HEPES, pH 7.2 with KOH). Store it at 4 °C. Warm it to room temperature before use.
3. Prepare Permeabilization Solution, which contains 0.005% saponin in Wash Solution. Prepare it fresh daily.
4. Prepare 0 Ca²⁺ Internal Solution (100 mM potassium acetate, 15 mM KCl, 0.35 mM EGTA, 0.75 mM MgCl₂, 10 mM HEPES, pH adjusted to 7.2 with KOH). Store it at 4 °C. Warm it to room temperature before use.
5. Prepare internal solution containing Ca²⁺. Add CaCl₂ to the internal solution above to reach the desired concentration of free Ca²⁺. The amount of CaCl₂ to add is calculated using the MaxChelator program (maxchelator.stanford.edu).
6. Prepare 1 mM Rhod-2/AM stock in DMSO and store it at -20 °C until use.
7. Prepare 1 mM MitoTracker green stock in DMSO and store it at -20 °C until use.

2. Plating Cells for Imaging

1. Wash 22 x 22 cm 2 glass coverslips with 100% ethanol and allow the coverslips to air dry.
2. Place the glass coverslips into individual wells of a 6-well tissue culture dish.
   NOTE: Coverslips can be coated with laminin, poly-L-lysine, or similar matrix to promote cell attachment.
3. Trypsinize and plate the cells onto the coverslips aiming for approximately 70% confluence on the day of imaging.

3. Loading Cells with the Rhod-2/AM and MitoTracker Green

1. Prepare Rhod-2/AM-MitoTracker green working solution. Add 20 μL of Rhod-2/AM 1 mM stock, 0.2 μL of MitoTracker green 1 mM stock, and 2.5 μL of 20% pluronic F-127 to 1 mL of Tyrode's solution. The final concentration of Rhod-2 in the working solution is 20 µM and the final concentration of MitoTracker green is 200 nM.
2. Gently remove the growth media from the coverslip.
   NOTE: A wash step is not necessary prior to dye solution addition.
3. Add the Rhod-2/AM-MitoTracker green solution in a dropwise fashion onto the coverslip until the coverslip is just covered (approximately 3-4 drops per coverslip).
4. Incubate it for 30 min at room temperature protected from light to allow the cells to load with the dyes.
5. De-esterify the Rhod-2/AM. Gently remove the Rhod-2/AM-MitoTracker green solution, replace it with fresh room temperature Tyrode's solution, and incubate it for 30 min at room temperature protected from light.

4. Confocal Imaging of the Mitochondrial Rhod-2/AM and MitoTracker Green Fluorescence

1. Transfer the coverslip to the microscope imaging chamber and fill the chamber with wash solution.
2. Under settings for observing cells in phase contrast at 40X, adjust focus until cells are visible and in focus.
3. Permeabilize the plasma membrane of Rhod-2/AM-MitoTracker green-loaded cells to remove cytosol-localized Rhod-2, while retaining mitochondria-localized dye.
   1. Remove the Wash Solution from the coverslip.
   2. Replace it with Permeabilization Solution for approximately 1 min.
   3. Visually monitor the plasma membrane morphology throughout the permeabilization process. Permeabilized cells will develop a roughened surface.
   4. When permeabilization is complete, immediately remove the Permeabilization Solution and replace it with 0 Ca²⁺ Internal Solution.
4. Simultaneously image Rhod-2 fluorescence (excitation using the 559 nm laser line and emission collected at wavelengths between 575-675 nm) and MitoTracker green fluorescence (excitation using the 488nm laser line and emission collected at wavelengths between 505-525 nm). Focus on permeabilized cells displaying a clear colocalization of Rhod-2 and MitoTracker green.
5. Decrease the microscope laser and gain settings such that the mitochondrial Rhod-2 fluorescence is dim and just visible.
6. Select microscope settings to acquire 2-dimensional scans at an appropriate frame rate and time course for the specific application. A frame rate of at least 30 frames/s is recommended to accurately capture the kinetics of changes in mitochondrial Ca²⁺.
7. Remove the 0 Ca²⁺ Internal Solution without disturbing the cells or microscope focus.
8. Start image acquisition.
9. Add Ca²⁺-replete Internal Solution at the 10 s time point either manually or with a perfusion system.
10. Select regions of interest (ROIs) to encompass regions of colocalization between mitochondrial Rhod-2 and MitoTracker green signal in the image acquisition software.
    NOTE: Arbitrary fluorescence (F) values for each time point of the recording are obtained for each ROI. These values are background subtracted and normalized to the initial fluorescence (prior to Ca²⁺ addition; F₀) and plotted as F/F₀ over time for data presentation and quantification of the amplitude of the change of mitochondrial Ca²⁺.