Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Summary

Mitochondria can utilize the electrochemical potential across their inner membrane (Δ_Ψm) to sequester calcium (Ca²⁺), allowing them to shape cytosolic Ca²⁺ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca²⁺ uptake and ΔΨ_m in live cells using fluorescent dyes and confocal microscopy.

Abstract

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca²⁺) buffers to tightly regulate intracellular Ca²⁺ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (ΔΨ_m) to sequester Ca²⁺. The influx of Ca²⁺ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains ΔΨ_m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.

We describe here a method for simultaneously measuring mitochondria Ca²⁺ uptake and ΔΨ_m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca²⁺ can be measured using the fluorescent Ca²⁺ indicator Fluo-4, AM, with measurement of ΔΨ_m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca²⁺ and ΔΨ_m simultaneously. Using the sequential addition of Ca²⁺ aliquots, mitochondrial Ca²⁺ uptake can be monitored, and the concentration at which Ca²⁺ induces mitochondrial membrane permeability transition and the loss of ΔΨ_m determined.

Introduction

Mitochondria play an important role in regulating intracellular Ca²⁺ concentration by acting as local Ca²⁺ buffers¹. Ca²⁺ enters the mitochondria via the Ca²⁺ uniporter, a process driven by the electrochemical gradient that exists across the mitochondrial inner membrane (ΔΨ_m)². Once inside the mitochondrial matrix, Ca²⁺ can activate oxidative phosphorylation by stimulating three rate-limiting dehydrogenases of the citric acid cycle³. This stimulation maintains ΔΨ_m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix. If the Ca²⁺ concentration within the mitochondria becomes very high, mitochondrial permeability transition can be initiated, resulting in the dissipation of ΔΨ_m, the cessation of oxidative phosphorylation and the induction of cell death signaling pathways⁴.

The important role that mitochondria play in the spatial buffering of cellular calcium makes the accurate monitoring of mitochondrial calcium critical. Various methods have been established to monitor mitochondrial calcium, including the use of rhodamine based dyes. One such dye, Rhod-2, AM, is quite effective at partitioning to the mitochondria to measure mitochondrial Ca²⁺ levels^5,6. However, care must be used as some dye will accumulate in other organelles, such as liposomes, or remain in the cell cytosol. Nevertheless, downstream analyses can be employed to distinguish these signals from those from the mitochondria⁷.

Another technique to monitor mitochondrial calcium utilizes fluorescent reporter constructs⁸. The benefit of these genetically encoded probes is that they can be specifically targeted to the mitochondria by using endogenous N-terminal peptides, for example the N-terminal targeting signal of human COX subunit VIII. This system has been employed to generate a mitochondrial-targeted aequorin probe which has proved extremely useful for investigating mitochondrial calcium signaling⁹. The main drawback of these genetically encoded probes is that they need to be introduced into the cells by transient expression (which is not feasible for certain cell types and can produce variable results) or by creating stable expression systems (which is time consuming).

To circumvent the problems outlined above, we have developed a new protocol to measure mitochondrial Ca²⁺ and ΔΨ_m simultaneously. This protocol is based on a previously described method that adds exogenous calcium to permeabilized cells¹⁰. Our protocol has three main advantages over other methods: firstly, we use Fluo-4, AM and TMRM to monitor mitochondrial Ca²⁺ and ΔΨ_m, two dyes that have very distinct spectral properties; secondly, the cells are permeabilized so that the Fluo-4 signal is only detecting mitochondrial Ca²⁺ and not Ca²⁺ localized to other organelles or the cell cytosol; and thirdly, the use of Fluo-4 to detect mitochondrial Ca²⁺ allows for fast and simple cell staining, negating any cell transfection or transformation issues that exist if using genetically encoded probes.

Protocol

1. Preparation of Cells

1. Grow cells on 10 cm cell culture dishes or 75 cm² flasks in culture media [10 ml Dulbecco's Modified Eagles Medium (DMEM) supplemented with 5% (v/v) fetal bovine serum (FBS) and 1x penicillin/streptomycin (p/s)] at 37 °C/5% CO₂.
2. To harvest cells, remove media by aspiration, then wash with 5 ml 1x phosphate buffered saline (1x PBS). Remove 1x PBS by aspiration, then add 1.5 ml 0.25% (w/v) Trypsin/0.25% (w/v) ethylenediaminetetraacetic acid (EDTA) and incubate at 37 °C/5% CO₂ for 2 min. Tap dish or flask gently to remove cells, then resuspend in 5 ml culture media.
3. Count cells by adding 12 µl of resuspended cells onto a hemocytometer.
4. Plate cells in dishes or chambered coverslips for confocal imaging.
   NOTE: These will have a suitable plastic or glass bottom that has been designed for use with inverted microscopes.
5. Plate cells so that ~80% confluency is achieved on the day of imaging. For example, approximately 2 x 10⁴ 143B osteosarcoma cells can be plated into one well of an 8-well chamber slide one day before imaging.
6. Incubate cells overnight at 37 °C/5% CO₂ to allow cells to attach and recover.

2. Buffers for TMRM and Fluo-4 Imaging

1. Prepare Record Solution (RS) containing 156 mM NaCl, 3 mM KCl, 2 mM MgSO₄, 1.25 mM KH₂PO₄, 10 mM D-glucose, 2 mM CaCl₂ and 10 mM HEPES pH 7.35. Store RS in aliquots at  -20 ºC.
2. Prepare Intracellular Medium (IM) containing 6 mM NaCl, 130 mM KCl, 7.8 mM MgCl₂, 1 mM KH₂PO₄, 0.4 mM CaCl₂, 2 mM EGTA, 10 mM HEDTA, 2 mM malate, 2 mM glutamate, 2 mM ADP and 20 mM HEPES pH 7.1. 200 mM stock solutions of malate, glutamate and ADP can be prepared separately, aliquoted, and stored at -20 °C. These stocks can be added to the IM just before use.
3. Ca²⁺ free Hank's buffered salt solution (HBSS) can be obtained pre-prepared from commercial suppliers. Add ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to a final concentration of 500 µM.
4. Prepare a stock solution of 10 mM tetramethylrhodamine, methyl ester, perchlorate (TMRM) in 100% methanol. From this stock, make a working stock of 2 µM in distilled H₂O.
5. Prepare a stock solution of 10 mM Verapamil in 100% ethanol.
6. Prepare a 1 mg/ml (w/v) stock solution of Fluo-4 acetoxymethyl ester (Fluo-4, AM) in dimethyl sulfoxide (DMSO).
   NOTE: Fluo-4, AM is a calcium indicator that exhibits increased fluorescence upon binding Ca²⁺. Fluo-4 is an analog of fluo-3, with the two chlorine substituents replaced by fluorines. This results in increased fluorescence excitation at 488 nm and higher fluorescence signal levels. The AM ester group results in an uncharged Fluo-4 molecule that can permeate cell membranes. Once inside the cell, the AM ester is cleaved by nonspecific esterases, resulting in a charged form of Fluo-4 that is trapped within the cell.
7. Prepare a stock solution of 1 mM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) in 100% ethanol.
8. Prepare a stock solution of 25 mg/ml (w/v) digitonin in distilled H₂O.
9. Prepare a stock solution of 100 µM thapsigargin in DMSO.
10. Prepare a stock solution of 40 mM calcium chloride (CaCl₂) in distilled H₂O.

3. Staining of Cells with TMRM and Fluo-4, AM

1. Prepare RS staining solution with 20 nM TMRM, 5 µg/ml (w/v) Fluo-4, AM and 0.005% surfactant such as Pluronic F-127 in RS.
   NOTE: This surfactant is a nonionic polyol that facilitates the solubilization of Fluo-4, AM. Note: 10 µM Verapamil can also be added to inhibit TMRM export by the plasma membrane multidrug transporter if it is expressed in the cell type being analyzed.
2. Remove culture media from cells by pipette and wash with 100 µl 1x PBS.
3. Remove 1x PBS by pipette and incubate cells in 250 µl RS staining solution for 45 min at room temperature.
   NOTE: Once the Fluo-4, AM enters the cell, the AM ester is cleaved by cellular esterases. Incubating at room temperature inhibits esterase activity, allowing Fluo-4, AM to enter the mitochondria.
4. Remove RS staining solution by pipette and wash cells in 100 µl Ca²⁺ free HBSS to remove excess Fluo-4, AM and TMRM.
5. Prepare IM imaging solution with 25 µg/ml (w/v) digitonin, 200 nM TMRM and 1 µM thapsigargin in IM.
   NOTE: The concentration of digitonin may need to be adjusted to ensure that permeabilization of the mitochondrial inner membrane does not occur. This can be determined empirically by assessing the intensity of the TMRM signal at different concentrations of digitonin.
6. Remove the Ca²⁺ free HBSS by pipette and add 300 µl of IM imaging solution to the cells. Leave cells to equilibrate at room temperature for 10 min. Cells are now ready for imaging with CaCl₂ additions.

4. Cell Imaging

1. Place dish or chambered coverslip with cells in IM imaging solution onto an inverted laser scanning confocal microscope.
2. Use setting on the microscope lasers for the excitation and emission spectra of TMRM and Fluo-4. For example, use 543 nm He-Ne and 473 nm argon laser lines to excite TMRM and Fluo-4 respectively. Use a low laser power of approximately 5% to minimize any photo-damage to the cells.
3. Set microscope to scan images every 25 sec. Scan cells for 10 min to establish baseline readings for the TMRM and Fluo-4 signals.
   NOTE: The Fluo-4 signal may be weak or undetectable at resting calcium concentrations.
4. Add 3 μl of 40 mM CaCl₂ stock solution directly to the cells in 300 μl of IM imaging solution (a 1:100 dilution) and mix gently with a pipette. Pause the image scanning while adding and mixing the CaCl₂ if necessary.
   NOTE: The final free Ca²⁺ ion concentration [Ca²⁺] in the IM imaging solution can be calculated using software such as Maxchelator WEBMAXC EXTENDED (http://www.stanford.edu/%7Ecpatton/maxc.html) or Chelator¹¹. A detailed description of how to calculate the final free Ca²⁺ ion concentration [Ca²⁺] is described in section 6 below.
5. Continue image scanning for approximately 4 min, then repeat CaCl₂ additions every 4 min until the desired TMRM or Fluo-4 signals are reached, usually around 8 to 10 additions.
6. Using a pipette, add a 1:100 dilution of 1 mM FCCP (final concentration of 10 μM) directly to the cells to dissipate ΔΨ_m. Image cells for a further 5 min. The experiment is now finished.

5. Image Analysis

1. Once imaging is complete, determine the intensity of the Fluo-4 and TMRM signals using image analysis software, for example ImageJ software with the Bio-Formats plugin (download 'bioformats_package.jar' from http://downloads.openmicroscopy.org/bio-formats/ and save it in the 'C:\Program Files\ImageJ\plugins' folder).
2. Open the imaging file (for example an .oif file) in ImageJ. Click on the selection tool and select a region of interest (ROI) that contains mitochondria. Use the initial TMRM signal to select the ROI.
3. Open 'Analyze>Tools>ROI Manager', click 'Add' to include the selected ROI for intensity measurement. Multiple ROIs can be selected on a single image for measurement. Repeat previous steps until all ROIs have been added to the ROI Manager.
4. Click 'More>Multi Measure’, make sure that ‘Measure all slices’ is selected and that ‘One Row Per Slice’ is deselected, then click ‘OK’ to obtain a table of mean fluorescent intensity of the TMRM and Fluo-4 signals for each ROI at each time point.
5. Use data from all ROIs to calculate the average intensities and standard deviation for the TMRM and Fluo-4 signals.

6. Calculating the Final Free Ca²⁺ Ion Concentration [Ca²⁺]

1. The free Ca²⁺ ion concentration [Ca²⁺] in the IM imaging solution can be determined using software such as Maxchelator WEBMAXC EXTENDED (http://www.stanford.edu/%7Ecpatton/maxc.html) or Chelator¹¹. For example, set the variables in WEBMAXC EXTENDED as follows: Temperature = 24 °C, pH = 7.1, Ionic strength = 0.162, EGTA = 0.002 M, HEDTA = 0.01 M, Ca²⁺ = 0.0004 M and Mg²⁺ = 0.0078 M. This results in a final free Ca²⁺ ion concentration [Ca²⁺] of 62 nM. Note that these programs cannot take into account every variable, such as reagent purity, accuracy of measurements and pH determination. The most accurate method for determining the free Ca²⁺ concentration is to use a calibrated Ca²⁺ selective electrode.

Results

We have used this protocol to examine the effects of an MT-ND5 mutation on the ability of 143B cell mitochondria to buffer increases in calcium¹². In the example shown here, control 143B cells were loaded with TMRM and Fluo-4, AM before permeabilization with digitonin. After 5 min of imaging, eight sequential additions of a 1:100 dilution of 40 mM exogenous CaCl₂ were made, with the final free Ca²⁺ ion concentration [Ca²⁺] ...

Discussion

Calcium plays a critical role in many cell processes, including muscle contraction, neuronal signaling and cell proliferation¹³. Increases in cell calcium concentrations are often associated with energy demand, with calcium able to directly stimulate mitochondrial oxidative phosphorylation to raise ATP generation³. It is therefore essential that we have the ability to effectively monitor mitochondrial calcium accumulation and to be able to compare how this function is affec...

Materials

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle Medium (DMEM)	ThermoFisher	10566016
fetal bovine serum (FBS)	ThermoFisher	16000044
1x phosphate buffered saline (PBS)	ThermoFisher	10010023
100x penicillin/streptomycin (p/s)	ThermoFisher	15140122
0.25% Trypsin / 0.25% EDTA	ThermoFisher	25200056
8-well chambered coverslip	ibidi	80826
NaCl	Sigma-Aldrich	793566
KCl	Sigma-Aldrich	P9541
MgSO4	Sigma-Aldrich	746452
KH2PO4	Sigma-Aldrich	795488
D-glucose	Sigma-Aldrich	G8270
CaCl2	Sigma-Aldrich	746495
HEPES	Sigma-Aldrich	H3375
MgCl2	Sigma-Aldrich	M2670
EGTA	Sigma-Aldrich	E4378
HEDTA	Sigma-Aldrich	H8126
malate	Sigma-Aldrich	M1000
glutamate	Sigma-Aldrich	G1626
ADP	Sigma-Aldrich	A5285
Ca2+ free Hank’s buffered salt solution (HBSS)	ThermoFisher	14175-095
tetramethylrhodamine, methyl ester, perchlorate (TMRM)	ThermoFisher	T668
Verapamil	Sigma-Aldrich	V4629
Fluo-4 acetoxymethyl ester (Fluo-4, AM)	ThermoFisher	F14201
dimethyl sulfoxide (DMSO)	ThermoFisher	D12345
carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)	Sigma-Aldrich	C2920
digitonin	Sigma-Aldrich	D141
thapsigargin	Sigma-Aldrich	T9033
Pluronic F-127	ThermoFisher	P3000MP
hemacytometer	VWR	631-0925
10 cm cell culture dishes	Corning	COR430167
75 cm2 cell culture flasks	Corning	COR430641