Overview
This video describes a confocal microscopy imaging-based assay to measure mitochondrial calcium uptake in cultured cells. The assay uses the calcium-sensitive fluorescent dye Rhod-2/AM to measure the mitochondrial calcium in permeabilized cultured cells.
Protocol
1. Reagents and Solutions
NOTE: The second part is experimental procedure for confocal imaging of mitochondrial Ca2+ in cultured cells
- 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.
- 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.
- Prepare Permeabilization Solution, which contains 0.005% saponin in Wash Solution. Prepare it fresh daily.
- 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.
- 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).
- Prepare 1 mM Rhod-2/AM stock in DMSO and store it at -20 °C until use.
- Prepare 1 mM MitoTracker green stock in DMSO and store it at -20 °C until use.
2. Plating Cells for Imaging
- Wash 22 x 22 cm 2 glass coverslips with 100% ethanol and allow the coverslips to air dry.
- 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. - 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
- 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.
- Gently remove the growth media from the coverslip.
NOTE: A wash step is not necessary prior to dye solution addition. - 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).
- Incubate it for 30 min at room temperature protected from light to allow the cells to load with the dyes.
- 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
- Transfer the coverslip to the microscope imaging chamber and fill the chamber with wash solution.
- Under settings for observing cells in phase contrast at 40X, adjust focus until cells are visible and in focus.
- Permeabilize the plasma membrane of Rhod-2/AM-MitoTracker green-loaded cells to remove cytosol-localized Rhod-2, while retaining mitochondria-localized dye.
- Remove the Wash Solution from the coverslip.
- Replace it with Permeabilization Solution for approximately 1 min.
- Visually monitor the plasma membrane morphology throughout the permeabilization process. Permeabilized cells will develop a roughened surface.
- When permeabilization is complete, immediately remove the Permeabilization Solution and replace it with 0 Ca2+ Internal Solution.
- 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.
- Decrease the microscope laser and gain settings such that the mitochondrial Rhod-2 fluorescence is dim and just visible.
- 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+.
- Remove the 0 Ca2+ Internal Solution without disturbing the cells or microscope focus.
- Start image acquisition.
- Add Ca2+-replete Internal Solution at the 10 s time point either manually or with a perfusion system.
- 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+.
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Materials
Name | Company | Catalog Number | Comments |
Olympus FV1000 Laser Scanning confocal microscope | Olympus | FV1000 | |
22 x 22 mm coverslips | Corning | 2850-22 | |
6-well plate | Corning | 3506 | |
MitoTracker green FM | Invitrogen | M7514 | |
Rhod-2, AM | Invitrogen | R1244 | |
DMSO | Invitrogen | D12345 | |
Pluronic F-127 | Invitrogen | P3000MP | |
HEPES | Sigma | H3375 | |
EGTA | Sigma | E8145 | |
Potassium chloride | Fisher | BP366-500 | |
Potassium phosphate monobasic | Sigma | P0662 | |
Magnesium chloride | Sigma | M2670 | |
Calcium chloride | Sigma | C4901 | |
Potassium acetate | Fisher | BP364-500 | |
Adenosine 5′-triphosphate magnesium salt | Sigma | A9187 | |
Phosphocreatine disodium salt | Sigma | P7936 | |
Saponin | Sigma | S7900 |