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>
	<li>Szabadkai, G., 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=Mitochondria:+the+hub+of+cellular+Ca2++signaling.">Mitochondria: the hub of cellular Ca2+ signaling.</a> Physiology. 23, 84-94 (2008).</li><li>Jacobson, J., 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=Interplay+between+mitochondria+and+cellular+calcium+signalling.">Interplay between mitochondria and cellular calcium signalling.</a> Mol. Cell. Biochem. 256-257, 209-218 (2004).</li><li>Bhosale, G., Sharpe, J. A., Sundier, S. Y., 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=Calcium+signaling+as+a+mediator+of+cell+energy+demand+and+a+trigger+to+cell.">Calcium signaling as a mediator of cell energy demand and a trigger to cell.</a> Ann. N. Y. Acad. Sci. 1350, 107-116 (2015).</li><li>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=Mitochondria+calcium-dependent+neuronal+death+and+neurodegenerative+disease.">Mitochondria calcium-dependent neuronal death and neurodegenerative disease.</a> Pflugers Arch. 464, 111-121 (2012).</li><li>Drummond, R. M., Mix, T. C., Tuft, R. A., Walsh, J. V., Fay, F. 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. W. . 2. n. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+cyclophilin+D+and+the+mitochondrial+permeability+transition+enhances+beta-cell+survival+and+prevents+diabetes+in+Pdx1+deficiency.">Targeting cyclophilin D and the mitochondrial permeability transition enhances beta-cell survival and prevents diabetes in Pdx1 deficiency.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 10214-10219 (2010).</li><li>Rao, V. K., Carlson, E. A., Yan, S. 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+permeability+transition+pore+is+a+potential+drug+target+for+neurodegeneration.">Mitochondrial permeability transition pore is a potential drug target for neurodegeneration.</a> Biochim. Biophys. Acta. 1842, 1267-1272 (2014).</li></ol>

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+ ...

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