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 acid (HEPES), 1 mM ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA), pH adjusted to 7.4 with KOH. Sterilize it through a 0.22 &#956;m filter and store it at 4 &#176;C. Ensure that the MS-EGTA buffer is pre-chilled to 4 &#176;C before use.</li>
	<li>Prepare 100 mL of KCl Buffer: 125 mM KCl, 20 mM HEPES, 1 mM KH2PO4, 2 mM MgCl2, 40 &#956;M EGTA and pH adjusted to 7.2 with KOH. Store it at room temperature.</li>
	<li>Prepare 1 mM calcium green-5N stock in dimethyl sulfoxide (DMSO). The calcium green-5N stock can be aliquotted and stored at -20 &#176;C.</li>
	<li>Prepare substrates for Ca2+ uptake.
	<ol>
		<li>Prepare 1 M sodium pyruvate, pH 7.4. Store it in aliquots at -20 &#176;C.</li>
		<li>Prepare 500 mM malate, pH 7.4. Store it in aliquots at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Isolation of Cardiac Mitochondria
<ol>
	<li>Euthanize the mouse according to institutional standards. 
	NOTE: For our institutionally approved method of euthanasia, mice were anesthetized by isofluorane inhalation and sacrificed by cervical dislocation.</li>
	<li>Collect the heart by opening chest cavity, cutting along either side of the ribs, flanking the heart, cutting away the diaphragm, and excising the heart tissue. 
	NOTE: In this protocol, mitochondrial isolation is performed from a whole heart collected from an adult mouse (approximately 120 mg), which should be sufficient for 3-4 experiments. For mitochondrial isolation from other mitochondria-rich tissues such as the liver, up to 200 mg of tissue can be used following the protocol described.</li>
	<li>Rinse the tissue thoroughly in 25 mL of ice-cold 1x phosphate buffered saline (PBS) ensuring that all blood is squeezed out of the ventricles. 
	NOTE: The heart will be sufficiently rinsed when the liquid squeezed out of the heart runs clear.</li>
	<li>Mince the heart into small pieces in 5 mL of ice-cold 1x PBS using a pair of sharp scissors.</li>
	<li>Discard the PBS and transfer minced heart tissue to a pre-chilled 7 mL glass-teflon dounce homogenizer with a 0.10 to 0.15 mm clearance.</li>
	<li>Add 5 mL of ice-cold MS-EGTA buffer, and homogenize the sample until tissue pieces are no longer visible (approximately 11 strokes). 
	NOTE: Do not over-homogenize the tissue as the goal is to obtain intact and functional mitochondria.</li>
	<li>Transfer the homogenate to a 15 mL tube.</li>
	<li>Centrifuge the homogenate at 600 x g at 4 &#176;C for 5 min to pellet nuclei and unbroken cells.</li>
	<li>Transfer the supernatant to a fresh 15 mL tube and centrifuge it at 10,000 x g at 4 &#176;C for 10 min to pellet mitochondria.</li>
	<li>Discard the supernatant and keep the mitochondrial pellet on ice.</li>
	<li>Wash the mitochondrial pellet twice using ice-cold MS-EGTA buffer. For each wash, resuspend the mitochondrial pellet in 5 mL of MS-EGTA buffer, centrifuge it at 10,000 x g at 4 &#176;C for 10 min, and discard the supernatant.</li>
	<li>After the final wash, discard the supernatant and resuspend the mitochondria in 100 &#956;L of ice cold MS-EGTA buffer. Keep the mitochondria on ice. 
	NOTE: Mitochondria should be used for experimentation within 1 h.</li>
	<li>Measure mitochondrial protein concentration using a Bradford Protein Assay26.</li>
</ol>
3. Plate Reader-based Measurement of Mitochondrial Calcium Uptake
NOTE: Here, it is described the protocol for analyzing mitochondrial Ca2+ uptake using a multimode plate reader fitted with injectors. Any plate reader with the capability of reading calcium green-5N fluorescence (excitation/emission of 506/532 nm) in a kinetic mode with automated reagent injectors to keep the reaction protected from light can be used.
<ol>
	<li>Program the plate reader to perform a kinetic read of calcium green-5N fluorescence with measurements taken every second for a total assay time of 1,000 s. Additionally, program the reagent injectors to dispense 5 &#956;L of CaCl2 solution at the 30 s, 150 s, 300 s, 480 s, and 690 s time points. 
	NOTE: The timing of the CaCl2 injections are user defined, and can be adjusted for according to experimental needs.</li>
	<li>Prime the reagent injectors with the CaCl2 solution to be used. 
	NOTE: The concentration of the CaCl2 solution is user defined, and can be adjusted in subsequent runs to titrate the amount of Ca2+ required to trigger MPTP opening.</li>
	<li>Add 200 &#956;g of mitochondria to an individual well of a 96 well plate.</li>
	<li>Add the appropriate volume of KCl buffer to the well such that the total volume of mitochondria and KCl buffer comes to 197 &#956;L.</li>
	<li>Add 1 &#956;L of 1 M pyruvate, and 1 &#956;L of 500 mM malate to the mitochondria mixture. Pipet gently to mix, and incubate the mitochondria with the substrates for 2 min at room temperature to allow mitochondria to become energized.</li>
	<li>Add 1 &#956;L of 1 mM calcium green-5N stock. Mix gently by pipetting. 
	NOTE: Protect the reaction from light following the addition of the calcium green-5N dye.</li>
	<li>Start the pre-programmed kinetic protocol and monitor calcium green-5N fluorescence.</li>
</ol>
4. Reagents and Solutions
NOTE: The second part is experimental procedure for confocal imaging of mitochondrial Ca2+ in cultured cells
<ol>
	<li>Make Tyrode&#39;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 &#176;C. Warm it to room temperature before use.</li>
	<li>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 &#176;C. Warm it to room temperature before use.</li>
	<li>Prepare Permeabilization Solution, which contains 0.005% saponin in Wash Solution. Prepare it fresh daily.</li>
	<li>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 &#176;C. Warm it to room temperature before use.</li>
	<li>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).</li>
	<li>Prepare 1 mM Rhod-2/AM stock in DMSO and store it at -20 &#176;C until use.</li>
	<li>Prepare 1 mM MitoTracker green stock in DMSO and store it at -20 &#176;C until use.</li>
</ol>
5. Plating Cells for Imaging
<ol>
	<li>Wash 22 x 22 cm2 glass coverslips with 100% ethanol and allow the coverslips to air dry.</li>
	<li>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.</li>
	<li>Trypsinize and plate the cells onto the coverslips aiming for approximately 70% confluence on the day of imaging.</li>
</ol>
6. Loading Cells with the Rhod-2/AM and MitoTracker Green
<ol>
	<li>Prepare Rhod-2/AM-MitoTracker green working solution. Add 20 &#956;L of Rhod-2/AM 1 mM stock, 0.2 &#956;L of MitoTracker green 1 mM stock, and 2.5 &#956;L of 20% pluronic F-127 to 1 mL of Tyrode&#39;s solution. The final concentration of Rhod-2 in the working solution is 20 &#181;M and the final concentration of MitoTracker green is 200 nM.</li>
	<li>Gently remove the growth media from the coverslip. 
	NOTE: A wash step is not necessary prior to dye solution addition.</li>
	<li>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).</li>
	<li>Incubate it for 30 min at room temperature protected from light to allow the cells to load with the dyes.</li>
	<li>De-esterify the Rhod-2/AM. Gently remove the Rhod-2/AM-MitoTracker green solution, replace it with fresh room temperature Tyrode&#39;s solution, and incubate it for 30 min at room temperature protected from light.</li>
</ol>
7. Confocal Imaging of the Mitochondrial Rhod-2/AM and MitoTracker Green Fluorescence
<ol>
	<li>Transfer the coverslip to the microscope imaging chamber and fill the chamber with wash solution.</li>
	<li>Under settings for observing cells in phase contrast at 40X, adjust focus until cells are visible and in focus.</li>
	<li>Permeabilize the plasma membrane of Rhod-2/AM-MitoTracker green loaded cells to remove cytosol-localized Rhod-2, while retaining mitochondria-localized dye.
	<ol>
		<li>Remove the Wash Solution from the coverslip.</li>
		<li>Replace it with Permeabilization Solution for approximately 1 min.</li>
		<li>Visually monitor the plasma membrane morphology throughout the permeabilization process. Permeabilized cells will develop a roughened surface.</li>
		<li>When permeabilization is complete, immediately remove the Permeabilization Solution and replace it with 0 Ca2+ Internal Solution.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Decrease the microscope laser and gain settings such that the mitochondrial Rhod-2 fluorescence is dim and just visible.</li>
	<li>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+.</li>
	<li>Remove the 0 Ca2+ Internal Solution without disturbing the cells or microscope focus.</li>
	<li>Start image acquisition.</li>
	<li>Add Ca2+-replete Internal Solution at the 10 s time point either manually or with a perfusion system.</li>
	<li>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+.</li>
</ol>

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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&amp;prime;-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...

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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 ...

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JoVE Journal

Biochemistry

13.8K Views.  Emory University School of Medicine. Hier presenteren we twee protocollen voor het meten van mitochondriale Ca2 + toestroom in geïsoleerde mitochondriën en gekweekte cellen. Voor geïsoleerde mitochondriën, detailleren wij een plaat lezer gebaseerde Ca2 + import assay met behulp van de Ca2 + gevoelige kleurstof calcium groen-5N. Voor gekweekte cellen beschrijven we een methode van de confocal microscopie met behulp van de Ca2 + kleurstof Rhod-2/AM.

Video: Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Biology

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in cytosolic Ca2+ buffering, and the ATP production in mitochondrial can be mediated by intramitochondrial free Ca2+ concentration. Ca2+ retention capacity can be regarded as the capability of mitochondria to retain calcium in the mitochondrial matrix. Accumulated intracellular Ca2+ leads to the permeability of the inner mitochondrial membrane, termed the opening of mitochondrial permeability transition pore (mPTP), which leads to the leakage of molecules with a molecular weight less than 1.5 kDa. Ca2+-triggered mitochondria swelling is used to indicate the mPTP opening. Here, we describe two assays to examine the Ca2+ retention capacity and Ca2+-triggered mitochondrial swelling in isolated mitochondria. After certain amounts of Ca2+ are added, all steps can be completed in one day and recorded by a microplate reader. Thus, these two simple and effective assays can be adopted to assess the Ca2+-related mitochondrial functions.

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

This protocol aims to describe a method to examine the Ca2+ retention capacity and Ca2+- triggered mitochondrial swelling of isolated mitochondria of SH-SY5Y cells step-by-step.

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in ...

Neuroscience

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs)

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of mitochondrial Ca2+ contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Here we present a cell-type specific and mitochondria targeting molecular approach for mitochondrial Ca2+ imaging in astrocytes and neurons in vitro and in vivo. We constructed DNA plasmids encoding mitochondria-targeting genetically encoded Ca2+ indicators (GECIs) GCaMP5G or GCaMP6s (GCaMP5G/6s) with astrocyte- and neuron-specific promoters gfaABC1D and CaMKII and mitochondria-targeting sequence (mito-). For in vitro mitochondrial Ca2+ imaging, the plasmids were transfected in cultured astrocytes and neurons to express GCaMP5G/6s. For in vivo mitochondrial Ca2+ imaging, adeno-associated viral vectors (AAVs) were prepared and injected into the mouse brains to express GCaMP5G/6s in mitochondria in astrocytes and neurons. Our approach provides a useful means to image mitochondrial Ca2+ dynamics in astrocytes and neurons to study the relationship between cytosolic and mitochondrial Ca2+ signaling, as well as astrocyte-neuron interactions.

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators GECIs

This proctocol aims to provide a method for in vitro and in vivo mitochondrial Ca2+ imaging in astrocytes and neurons.

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

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.

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+ ...

Calcium Influx Assay to Measure Mitochondrial Calcium Uptake in Cultured Cells

Source: Maxwell, J.T., et al., <a href="https://app.jove.com/v/57225">Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells.</a> J. Vis. Exp. (2018).
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.

Source: Maxwell, J.T., et al., Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells. J. Vis. Exp. (2018).
This video ...

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Biological Techniques

Assay Techniques

Cell-based assays

Mitochondrial Calcium Uptake Assay: A Plate Reader-Based Method for Measuring Calcium Uptake in Isolated Mitochondria

Source: Maxwell, J.T., et al., <a href="https://app.jove.com/v/57225">Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells.</a> J. Vis. Exp. (2018).
This video describes a plate reader-based assay to measure mitochondrial calcium uptake in isolated mitochondria. The assay analyzes the fluorescence signal of a calcium-sensitive fluorescent dye to study the kinetics of mitochondrial calcium uptake and calcium overload.

Biochemical assays

Determination of the Calcium Retention Capacity of Isolated Mitochondria

Source: Li, W., et. al. <a href="https://app.jove.com/v/56236">Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay.</a> J. Vis. Exp. (2018)
This video demonstrates the procedure to assess mitochondrial calcium retention capacity by monitoring real-time changes in fluorescence as mitochondria uptake calcium. This assay is useful for studying mitochondrial function and calcium-related processes, including apoptosis and neurodegenerative diseases.

Source: Li, W., et. al. Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay. J. Vis. Exp. (2018)
This video ...

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

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