This work was supported by grant funding from the American Heart Association (J.Q.K.).

All methods described in this protocol have been approved by the Institutional Animal Care and Use Committee of Emory University.
NOTE: The first part is the experimental procedure for measuring mitochondrial Ca2+ influx in isolated cardiac mitochondria using a plate reader.
1. Reagents and Solutions
<ol>
	<li>Make 500 mL of MS-EGTA buffer for mitochondrial isolation: 225 mM mannitol, 75 mM sucrose, 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic aci...

<ol>
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A., Rizzuto, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+mitochondrial+ATP+synthesis+by+calcium:+evidence+for+a+long-term+metabolic+priming.">Regulation of mitochondrial ATP synthesis by calcium: evidence for a long-term metabolic priming.</a> P Natl Acad Sci USA. 96 (24), 13807-13812 (1999).</li><li>Haworth, R. A., Hunter, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ca2+-induced+membrane+transition+in+mitochondria.+II.+Nature+of+the+Ca2++trigger+site.">The Ca2+-induced membrane transition in mitochondria. II. Nature of the Ca2+ trigger site.</a> Arch Biochem Biophys. 195 (2), 460-467 (1979).</li><li>Hunter, D. R., Haworth, R. 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The authors have no disclosures to report.

Here, we describe two different approaches to measure mitochondrial Ca2+ influx. The plate reader-based calcium green-5N method monitors extramitochondrial Ca2+ levels and is a Ca2+ uptake assay that is well suited for measurements in isolated mitochondria. While we have shown representative results from isolated murine cardiac mitochondria, this assay can be readily adapted for mitochondria isolated from tissues with high mitochondrial abundance including the liver, skeletal muscle, and ...

Mitochondria are critical sites of intracellular Ca2+ storage and signaling. Decades of research have demonstrated that mitochondria have the ability to import and sequester Ca2+ 1,2. Mitochondria, however, are not merely passive sites of Ca2+ storage. Ca2+ at the mitochondrial compartment performs fundamental signaling functions including regulation of metabolic output and activation of mitochondrial-mediated cell death pathways, which has been reviewed previously3. For metabolic regulation, Ca2+ enhances the activity of three matrix-localized dehydrogenases of the tricarboxylic acid cycle as well as respiratory chain complexes, to increase mitochondrial energy production4,5. With mitochondrial Ca2+ overload and dysregulated mitochondrial Ca2+ handling, Ca2+ triggers mitochondrial permeability transition pore (MPTP) opening, leading to mitochondrial inner membrane permeabilization, membrane potential loss, mitochondrial dysfunction, swelling, rupture and ultimately, cell death6,7,8,9. Thus, mitochondrial Ca2+ signaling directly impacts both cellular life and death pathways through metabolic control and MPTP-death axis.
In recent years, there has been rapidly expanding interest in the study of mitochondrial Ca2+ dynamics due in large part to the identification of the molecular constituents of the mitochondrial Ca2+ uniporter complex, a mitochondrial inner membrane transporter that is a primary mode of Ca2+ import into the mitochondrial matrix 10,11,12. Identification of these structural and regulatory subunits of the uniporter has brought forth the possibility of genetically targeting mitochondrial Ca2+ influx to modulate mitochondrial function and dysfunction and facilitated the study of the contribution of the uniporter complex and mitochondrial Ca2+ influx to disease13,14,15. Indeed, mitochondrial Ca2+ signaling has been implicated in the pathologies of a diverse array of diseases ranging from cardiac disease to neurodegeneration, and cancer16,17,18,19,20.
Given the fundamental importance of mitochondrial Ca2+ signaling in metabolism and cell death, and combined with the broad reach of biological systems that mitochondrial Ca2+ signaling impacts, methods to assess mitochondrial Ca2+ influx are of great interest. Not surprisingly, a variety of techniques and tools to measure mitochondrial Ca2+ have been developed. These include methods that utilize tools such as fluorescent Ca2+-sensitive dyes21,22 and genetically-encoded Ca2+ sensors targeted to the mitochondria, such as cameleon and aequorin23,24. The goal of this article is to highlight different methods and model systems in which mitochondrial Ca2+ uptake can be measured. We present two experimental methods to assess mitochondrial Ca2+ influx capacity. Using cardiac mitochondria as an example, we detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N that is ideally suited for isolated tissue mitochondria14. Using cultured NIH 3T3 cells, we also describe a confocal microscopy imaging-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM25.

<table>
	<tbody>
		<tr>
			<td>Olympus FV1000 Laser Scanning confocal microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy Neo2 Multimode microplate reader with injectors</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Homogenizer</td>
			<td>Kimble</td>
			<td>886000-0022</td>
			<td></td>
		</tr>
		<tr>
			<td>22 x 22 mm coverslips</td>
			<td>Corning</td>
			<td>2850-22</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>Corning</td>
			<td>3628</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Green-5N</td>
			<td>Invitrogen</td>
			<td>C3737</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker green FM</td>
			<td>Invitrogen</td>
			<td>M7514</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhod-2, AM</td>
			<td>Invitrogen</td>
			<td>R1244</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Invitrogen</td>
			<td>D12345</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Invitrogen</td>
			<td>P3000MP</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Mannitol</td>
			<td>Sigma</td>
			<td>M9546</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>EMD Millipore</td>
			<td>8510</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E8145</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Fisher</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma</td>
			<td>P2256</td>
			<td></td>
		</tr>
		<tr>
			<td>L-malic acid</td>
			<td>Sigma</td>
			<td>M1125</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium acetate</td>
			<td>Fisher</td>
			<td>BP364-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-triphosphate magnesium salt</td>
			<td>Sigma</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphocreatine disodium salt</td>
			<td>Sigma</td>
			<td>P7936</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma</td>
			<td>S7900</td>
			<td></td>
		</tr>
		<tr>
			<td>Ru360</td>
			<td>Calbiochem</td>
			<td>557440</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyses of mitochondrial calcium influx in isolated mitochondria and cultured cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Figure 1 shows mitochondrial Ca2+ uptake measurements in isolated cardiac mitochondria using the plate reader-based platform and the Ca2+ dye calcium green-5N. Under control conditions (Figure 1A), cardiac mitochondria were suspended in KCl buffer containing calcium green-5N and then challenged with sequential pulses of CaCl2 (5 &#956;L of a 0.6 mM CaCl2 solution) added at the 30 s, 150...

Watch this Scientific Journal Video about Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells at JoVE.com

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

The overall goal of this procedure is to measure mitochondrial calcium influx in isolated mitochondria and cultured cells. This method can help answer key questions in the calcium signaling field such as how mitochondria help to shape the cytosolic calcium landscape, and the role of mitochondrial calcium dynamics in disease conditions. The main advantage of this technique is that our protocols can be adapted to a variety of model systems to assess mitochondrial calcium influx capacity.I will be demonstrating the plate-reader-based mitochondrial calcium uptake procedure. And Dr.Joshua Maxwell, an instructor in the Department of Pediatrics will be demonstrating the confocal-microscope-based procedure to measure mitochondrial calcium influx. Start this experiment by thoroughly rinsing previously isolated heart in 25 milliliters of ice-cold one-X PBS making sure that the blood is completely squeezed out of the ventricles.In five milliliters of ice-cold one-X PBS, using a pair of sharp scissors, mince the heart tissue into small pieces. Carefully discard the PBS and transfer the tissue to a pre-chilled seven-milliliter glass Teflon Dounce homogenizer. Then, add five milliliters of ice-cold MSEGTA buffer and homogenize until there are no visible tissue pieces.Transfer the homogenate to a 15-milliliter tube, and then centrifuge at 600 times G at four degrees Celsius for five minutes to pellet unwanted nuclei and unbroken cells. Transfer the supernatant to a fresh 15-milliliter tube, and centrifuge at 10, 000 times G at four degrees Celsius for 10 minutes. After discarding the supernatant, keep the mitochondrial pellet on ice.To wash the pellet, add five milliliters of ice-cold MSEGTA buffer to it, resuspend and then centrifuge at 10, 000 times G at four degrees Celsius for 10 minutes. After discarding the supernatant, repeat the wash and keep the pellet on ice. To start mitochondrial calcium uptake measurement using a plate reader, program the reader to perform a kinetic read of Calcium Green-5N florescence every second for a total time of 1, 000 seconds.Program the reagent injectors to dispense five microliters of calcium chloride solution at various time points. Then, prime the injectors with the calcium chloride solution that will be used. Next, add 200 micrograms of isolated mitochondria to one well of a 96-well plate and add the appropriate volume of potassium chloride buffer to achieve a total volume of 197 microliters.Then, add one microliter of one molar pyruvate, one microliter of 500-millimolar malate and one microliter of one-millimolar Calcium Green-5N stock and mix gently by pipetting. Incubate protected from light at room temperature for two minutes for mitochondria to become energized. Finally, start the pre-programmed kinetic protocol and monitor Calcium Green-5N florescence.For this part of the protocol, prepare Rhod-2 AM MitoTracker Green working solution by mixing 20 microliters of Rhod-2 AM, 0.2 microliters of MitoTracker Green, 2.5 microliters of 20%pluronic F-127 and one milliliter of Tyrode solution. Onto a previously-prepared coverslip, plated with NIH/3T3 cells, add the Rhod-2 AM MitoTracker Green solution drop-wise until covered. For the cells to load with the dyes, incubate the coverslip protected from light for 30 minutes at room temperature.To deesterify Rhod-2 AM, gently remove the Rhod-2 AM MitoTracker Green solution, and replace it with approximately 300 microliters of fresh Tyrode solution to cover the cells. Incubate the coverslip protected from light for 30 minutes at room temperature. Now, transfer the coverslip to the microscope imaging chamber and fill the chamber with wash solution.Adjust the focus to observe the cells and phase contrast at 40X magnification. To permeabilize the plasma membrane of Rhod-2 AM MitoTracker Green loaded cells, remove the wash solution from the coverslip and replace it with approximately 300 microliters of permeabilization solution to cover the cells. Monitor the plasma membrane morphology during this process.When permeabilized, cells will develop a roughened surface. Remove the permeabilization solution immediately after complete permeabilization and replace it with zero-calcium internal solution. Next, focus on permeabilized cells displaying a clear co-localization of Rhod-2 and MitoTracker Green to image Rhod-2 and Mitotracker Green florescence simultaneously.Next, decrease the microscope laser and gain settings to make the mitochondrial Rhod-2 florescence dim and barely visible. To capture the kinetics of mitochondrial calcium changes, select microscope settings to acquire two-dimensional scans at an appropriate frame-rate and time course. Remove the zero-calcium internal solution making sure not to disturb the cells and microscope focus and then start image acquisition.Using a syringe, manually add calcium-replete internal solution at the 10-seconds time point. Finally, in the image acquisition software, select regions of interest to encompass regions of co-localization of Rhod-2 and MitoTracker Green signal. When calcium chloride is added to isolated cardiac mitochondria, florescence from the calcium dye increases as mitochondria uptake calcium from the buffer florescence decreases.However, at the third edition of calcium, florescence suddenly starts increasing after a drop indicating mitochondrial calcium overload and mitochondrial permeability transition pore opening which can lead to mitochondrial inner membrane permeabilization. On the other hand, when mitochondria are pre-treated with an inhibitor of mitochondrial calcium uptake, there is an increase in florescence following each calcium addition. In cultured NIH/3T3 cells, the mitochondrial network is stained with MitoTracker Green dye.Cells are also stained with Rhod-2 dye and after saponin permeabilization, cytosolic Rhod-2 is washed away leaving just mitochondrial staining which co-localizes with the MitoTracker. Since Rhod-2 can accumulate in non-mitochondrial structures, the analysis should focus only on regions where MitoTracker and Rhod-2 co-localize. The addition of calcium causes an increase in Rhod-2 florescence in control cells whereas in cells treated with the inhibitor of mitochondrial calcium uptake, there is no change in florescence.These protocols can be applied to measure mitochondrial calcium uptake in many cell types including primary cells such as cardiac myocytes and neurons in immortalized cell lines as well as isolated mitochondria from many tissue types including heart, liver and brain. Calcium handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in different cells. The ability to accurately measure the influx of calcium from mitochondria is an important part of determining the role of mitochondrial calcium handling in these processes.After watching this video, you should have a good understanding of how to measure mitochondrial calcium influx in isolated mitochondria and cultured cells.

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Research

JoVE Journal

Biochemistry

13.6K visualizações.  Emory University School of Medicine. The overall goal of this procedure is to measure mitochondrial calcium influx in isolated mitochondria and cultured cells. This method can help answer key questions in the calcium signaling field such as how mitochondria help to shape the cytosolic calcium landscape, and the role of mitochondrial calcium dynamics in disease conditions. The main advantage of this technique is that our protocols can be adapted to a variety of model systems to assess mitochondrial calcium influx capacity....