Name: simultaneous measurement of mitochondrial calcium and mitochondrial membrane potential in live cells by fluorescent microscopy
Uploaded: 2017-01-24T14:00:00
Description: Watch this Scientific Journal Video about Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy at JoVE.com

We thank Dr Kirstin Elgass and Dr Sarah Creed from Monash Micro Imaging for technical assistance, and the Wellcome Trust and Medical Research Council UK for financial support. MMcK is supported the Australian Research Council Future Fellowship Scheme (FT120100459), the William Buckland Foundation, The Australian Mitochondrial Disease Foundation (AMDF), The Hudson Institute of Medical Research and Monash University. This work was supported by the Victorian Government Operational Infrastructure Support Scheme.

1. Preparation of Cells
<ol>
	<li>Grow cells on 10 cm cell culture dishes or 75 cm2 flasks in culture media [10 ml Dulbecco&#39;s Modified Eagles Medium (DMEM) supplemented with 5% (v/v) fetal bovine serum (FBS) and 1x penicillin/streptomycin (p/s)] at 37 &#176;C/5% CO2.</li>
	<li>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)...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Ca2++homeostasis+during+Ca2++influx+and+Ca2++release+in+gastric+myocytes+from+Bufo+marinus.">Mitochondrial Ca2+ homeostasis during Ca2+ influx and Ca2+ release in gastric myocytes from Bufo marinus.</a> J. Physiol. 522, 375-390 (2000).</li><li>Hajnoczky, G., Robb-Gaspers, L. D., Seitz, M. B., Thomas, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+of+cytosolic+calcium+oscillations+in+the+mitochondria.">Decoding of cytosolic calcium oscillations in the mitochondria.</a> Cell. 82, 415-424 (1995).</li><li>Davidson, S. M., Duchen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+mitochondrial+calcium+signalling+with+fluorescent+probes+and+single+or+two+photon+confocal+microscopy.">Imaging mitochondrial calcium signalling with fluorescent probes and single or two photon confocal microscopy.</a> Methods Mol. Biol. 810, 219-234 (2012).</li><li>Pozzan, T., Rudolf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+mitochondrial+calcium+in+vivo.">Measurements of mitochondrial calcium in vivo.</a> Biochim. Biophys. Acta. 1787, 1317-1323 (2009).</li><li>Rizzuto, R., Simpson, A. W., Brini, M., Pozzan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+changes+of+mitochondrial+Ca2++revealed+by+specifically+targeted+recombinant+aequorin.">Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin.</a> Nature. 358, 325-327 (1992).</li><li>Pitter, J. G., Maechler, P., Wollheim, C. B., Spat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+respond+to+Ca2++already+in+the+submicromolar+range:+correlation+with+redox+state.">Mitochondria respond to Ca2+ already in the submicromolar range: correlation with redox state.</a> Cell Calcium. 31, 97-104 (2002).</li><li>Schoenmakers, T. J., Visser, G. J., Flik, G., Theuvenet, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CHELATOR:+an+improved+method+for+computing+metal+ion+concentrations+in+physiological+solutions.">CHELATOR: an improved method for computing metal ion concentrations in physiological solutions.</a> BioTechniques. 12, 870-879 (1992).</li><li>McKenzie, M., Duchen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+Cellular+Bioenergetics+Causes+Mitochondrial+Calcium+Handling+Defects+in+MT-ND5+Mutant+Cybrids.">Impaired Cellular Bioenergetics Causes Mitochondrial Calcium Handling Defects in MT-ND5 Mutant Cybrids.</a> PLoS One. 11, e0154371 (2016).</li><li>Berridge, M. J., Lipp, P., Bootman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+versatility+and+universality+of+calcium+signalling.">The versatility and universality of calcium signalling.</a> Nat. Rev. Mol. Cell Biol. 1, 11-21 (2000).</li><li>Homolya, L., Hollo, Z., Germann, U. A., Pastan, I., Gottesman, M. M., Sarkadi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+cellular+indicators+are+extruded+by+the+multidrug+resistance+protein.">Fluorescent cellular indicators are extruded by the multidrug resistance protein.</a> J. Biol. Chem. 268, 21493-21496 (1993).</li><li>Fujimoto, K., Chen, Y., Polonsky, K. S., Dorn, G. 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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...

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 (&#916;&#936;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 &#916;&#936;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 &#916;&#936;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+ levels5,6. 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 &#916;&#936;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 &#916;&#936;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>ThermoFisher</td>
			<td>10566016</td>
		</tr>
		<tr>
			<td>fetal bovine serum (FBS)</td>
			<td>ThermoFisher</td>
			<td>16000044</td>
		</tr>
		<tr>
			<td>1x phosphate buffered saline (PBS)</td>
			<td>ThermoFisher</td>
			<td>10010023</td>
		</tr>
		<tr>
			<td>100x penicillin/streptomycin (p/s)</td>
			<td>ThermoFisher</td>
			<td>15140122</td>
		</tr>
		<tr>
			<td>0.25% Trypsin / 0.25% EDTA</td>
			<td>ThermoFisher</td>
			<td>25200056</td>
		</tr>
		<tr>
			<td>8-well chambered coverslip</td>
			<td>ibidi</td>
			<td>80826</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>793566</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541&#160;</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>746452</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>795488</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270&#160;</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>746495</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375&#160;</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M2670&#160;</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E4378&#160;</td>
		</tr>
		<tr>
			<td>HEDTA</td>
			<td>Sigma-Aldrich</td>
			<td>H8126&#160;</td>
		</tr>
		<tr>
			<td>malate</td>
			<td>Sigma-Aldrich</td>
			<td>M1000</td>
		</tr>
		<tr>
			<td>glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>G1626</td>
		</tr>
		<tr>
			<td>ADP</td>
			<td>Sigma-Aldrich</td>
			<td>A5285</td>
		</tr>
		<tr>
			<td>Ca2+ free Hank&#8217;s buffered salt solution (HBSS)&#160;</td>
			<td>ThermoFisher</td>
			<td>14175-095</td>
		</tr>
		<tr>
			<td>tetramethylrhodamine, methyl ester, perchlorate (TMRM)</td>
			<td>ThermoFisher</td>
			<td>T668</td>
		</tr>
		<tr>
			<td>Verapamil</td>
			<td>Sigma-Aldrich</td>
			<td>V4629</td>
		</tr>
		<tr>
			<td>Fluo-4 acetoxymethyl ester (Fluo-4, AM)</td>
			<td>ThermoFisher</td>
			<td>F14201</td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide (DMSO)</td>
			<td>ThermoFisher</td>
			<td>D12345</td>
		</tr>
		<tr>
			<td>carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)</td>
			<td>Sigma-Aldrich</td>
			<td>C2920</td>
		</tr>
		<tr>
			<td>digitonin</td>
			<td>Sigma-Aldrich</td>
			<td>D141</td>
		</tr>
		<tr>
			<td>thapsigargin</td>
			<td>Sigma-Aldrich</td>
			<td>T9033</td>
		</tr>
		<tr>
			<td>Pluronic F-127&#160;</td>
			<td>ThermoFisher</td>
			<td>P3000MP&#160;</td>
		</tr>
		<tr>
			<td>hemacytometer</td>
			<td>VWR</td>
			<td>631-0925</td>
		</tr>
		<tr>
			<td>10 cm cell culture dishes</td>
			<td>Corning</td>
			<td>COR430167</td>
		</tr>
		<tr>
			<td>75 cm2 cell culture flasks</td>
			<td>Corning</td>
			<td>COR430641</td>
		</tr>
	</tbody>
</table>

simultaneous measurement of mitochondrial calcium and mitochondrial membrane potential in live cells by fluorescent microscopy

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 (&#916;&#936;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 &#916;&#936;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 &#916;&#936;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 &#916;&#936;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 &#916;&#936;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 &#916;&#936;m determined.

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+] ...

Watch this Scientific Journal Video about Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy at JoVE.com

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

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) 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 &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

The overall goal of this procedure is to provide a simple method for simultaneously measuring mitochondrial calcium uptake and mitochondrial membrane potential in live cells using confocal microscopy. This method can help to answer key questions in the mitochondrial field, such as how mitochondria act as local calcium stores to tightly regulate intracellular calcium concentration. The main advantage of this technique is that both mitochondrial calcium and mitochondrial membrane potential can be specifically measured simultaneously in life cells without the need for genetic manipulation.Though this method can provide insight into calcium regulation in mitochondrial disease pathology, it can also be used to study other newer degenerative conditions, when urinal calcium regulation in crucial. In this procedure, grow the cells on ten centimeter cell culture dishes, or 75 square centimeter flasks, in culture media at 37 degrees Celsius for 24 hours. To harvest the cells, remove media by aspiration, and wash the cells with five milliliters of PBS.Afterward, remove PBS by aspiration. Then add 1.5 milliliters of Trypsin EDTA, and incubate the cells at 37 degrees Celsius for 2 minutes. Next tap the dish gently to remove the cells.Resuspend them in five milliliters of culture media. Afterward, count the cells with a hemocytometer. The day before confocal imaging, plate the cells at 60 to 70 percent confluency in dishes or chambered cover slips.Then incubate the cells overnight at 37 degrees Celsius to allow the cells to attach and recover. In this procedure, prepare the record staining solution with TMRM, Fluo 4 AM, and surfactant. Remove the culture media and wash the cells with 100 microliters of PBS.Next remove PBS and incubate the cells in 100 microliters of record staining solution for 45 minutes at room temperature. Following that, remove the record staining solution and wash the cells in 100 microliters of calcium-free HBSS to remove excess Fluo 4 AM and TMRM. Then prepare the intracellular imagining solution with digitonin, TMRM, and thapsigargin.Next remove the calcium-free HPSS and add 300 microliters of IM imaging solution to the cells. Leave the cells to equilibrate at room temperature for ten minutes before imaging with the calcium chloride additions. Use a low laser power of approximately 5 percent to minimize any photo damage to the cells.Use the setting on the microscope lasers for the excitation and emission spectra of TMRM and Fluo 4 AM.Use a low laser power of approximately five percent with high gain to minimize photo damage of the cells. Next set the microscope to scan images every twenty five seconds. Scan the cells for ten minutes to establish the base line readings for TMRM and Fluo 4 signals.Then pause the image scanning and add 3 microliters of 40 millimolar calcium chloride stock solution directly to the cells in 300 microliters of IM imaging solution. Mix gently with a pipette. When adding calcium solution to the cells, be careful not to move the dish on the microscope stage.Continue image scanning for approximately four minutes. Then repeat calcium chloride additions every four minutes until the desired TMRM or Fluo 4 signals are reached, usually around eight to ten additions. Subsequently add a 1 to 100 dilution of one millimolar FCCP directly to the cells to dissipate the inner membrane potential.Then image the cells for a further five minutes before the experiment is finished. Once the imaging is complete, determine the intensity of the Fluo 4 and TMRM signals using the image analysis software. To do so, open the image, click on the selection tool and select a region of interest that contains mitochondria.Multiple regions of interest can be selected for measurement by opening Analyze, Tools, ROI Manager. Click Add to include the selected ROI for intensity measurement. Repeat previous steps until all ROIs have been added to the ROI manager.Measure the average fluorescent intensity of the TMRM and Fluo 4 signals for each region of interest by selecting the more multi measure function. Make sure that the Measure All Slices is selected, and that One Row Per Slice is deselected. Then click Okay to obtain a table of mean fluorescent intensity of the TMRM and Fluo 4 signals for each ROI at each time point.Then combine the data to calculate the average intensity and standard deviation for the TMRM and Fluo 4 signals. In this experiment, 143B cells were preloaded with Fluo 4 AM and TMRM before permeabilization with digitonin. Following each calcium chloride addition, the TMRM signal decreases as the influx of calcium ions into the mitochondria dissipates the mitochondrial membrane potential.A small membrane potential is evident following permeability transition as the addition of 10 micromolar FCCP still induces a collapse of membrane potential. Here the mitochondrial calcium concentration, indicated by Fluo 4 signal, begins to increase following the second calcium chloride addition when the free calcium in the media reaches 0.32 micromolar. Each subsequent calcium chloride addition caused a progressive increase in mitochondrial calcium.Shown here are the images of simultaneous measurements of membrane potential in mitochondrial calcium. The red signal indicates TMRM and the green signal indicates Fluo 4. Once mastered, this technique can be done in approximately two hours if performed properly.After watching this video, you should have a good understanding of how to measure mitochondrial calcium levels and mitochondrial membrane potential simultaneously on confocal microscopy using the addition of exogenous calcium to the permeabilized cells. Don't forget that working with FCCP and digitonin can be extremely hazardous, and precautions such as wearing PPE should always been taking when performing this procedure.

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