This study was supported by grants from grant of the Outstanding Scientist of Shandong (JQ201421) and grant from NSFC (81371226).

1. Isolation of Mitochondria
NOTE: All solutions and equipment should be precooled to 0 - 4 &#176;C and kept on ice.
<ol>
	<li>Seed about 3 x 106 SH-SY5Y cells per 10 cm cell culture dish. Culture cells in high-glucose Dulbecco&#39;s modified Eagle medium with 10% fetal bovine serum, penicillin (100 U/mL)-streptomycin (100 &#181;g/mL). Maintain at 37 &#176;C in an incubator containing 5% CO2 overnight. 
	NOTE: At least 1 - 2 x 107 SH-SY5Y cells are needed for each isolation assay.</li>
	<li>Remove the medium and wash the cells with about 1 mL of ice cold PBS in a 10 cm cell culture dish 3 times.</li>
	<li>Add new 1 mL of ice cold PBS into 10 cm cell culture dish and scrape the adherent cells into a new 1.5 mL tube. Centrifuge at 800 x g for 10 min at 4 &#176;C.</li>
	<li>During the centrifugation, prepare 5 mL mitochondrial isolation buffer (250 mM sucrose, 10 mM HEPES(4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA) supplemented with 50 &#956;L of protease inhibitors stock solution (100x&#160;concentration; 1 EDTA-free tablet dissolved in 500 &#181;L of ddH2O).</li>
	<li>Remove the supernatant and resuspend the pellet with 1 mL of mitochondrial isolation buffer. Incubate the 1.5 mL tube on ice for 30 min.</li>
	<li>Lyse the suspended cells with a glass homogenizer (15 - 25 strokes) up and down manually on ice. 
	NOTE: Make sure there are no bubbles when cells are homogenized. Count the intact cells and bared nuclei by microscope visually and confirm that &#62;90% cell breakage has occurred.</li>
	<li>Transfer the homogenate into a 1.5 mL tube and centrifuge at 1,000 x g for 5 min at 4 &#176;C to remove the debris (nuclei and remaining intact cells).</li>
	<li>Transfer the supernatant into a new 1.5 mL tube and centrifuge at 1,000 x g for 5 min 4 &#176;C.</li>
	<li>Transfer the supernatant to a new tube and centrifuge at 14,000 x g for 15 min 4 &#176;C.</li>
	<li>Remove the supernatant and resuspend the pellet containing mitochondria with 1 mL of mitochondrial isolation buffer.</li>
	<li>Centrifuge the solution for 15 min at 14,000 x g 4 &#176;C to precipitate the mitochondria.</li>
	<li>The resulting mitochondria pellet must be kept on ice and resuspended in 50 - 100 &#956;L KCl media (125 mM KCl, 2 mM K2HPO4, 1 mM MgCl2, 20 mM HEPES, 5 mM glutamate, 5 mM malate, and 2 &#956;M rotenone, pH 7.4) according to the pellet volume before any assay.</li>
	<li>Use 5 &#956;L of mitochondrial solution to measure the mitochondrial protein concentration by standard BCA assay, measuring OD562 using a plate reader13.</li>
</ol>
2. Determination of Mitochondrial Ca2+ Retention Capacity
<ol>
	<li>Prepare the KCl media and add the Ca2+-binding green fluorescent dye to a final concentration of 0.5 &#956;M before the experiment.</li>
	<li>Transfer 1 mL of isolated mitochondria (0.4 mg/mL) in KCl media containing 0.5 &#956;M Ca2+-binding green fluorescent dye into each 6-well plate well.</li>
	<li>Incubate the mitochondria and the Ca2+-binding green fluorescent dye in the 6-well plate at room temperature protected from ambient light for 1 min. Add 4 &#956;L aliquots of a 20 mM CaCl2 solution (20 mM CaCl2, 127 mM KCl, 1 mM MgCl2, 20 mM HEPES, 5 mM glutamate, 5 mM malate, pH 7.4) to each 6-well plate well using the automatic dispense setting to introduce 200 nmol Ca2+/mg mitochondrial protein.</li>
	<li>Use a fluorescence spectrometer to monitor the fluorescence changes every 3 s for 2 min with an excitation wavelength of 506 nm and an emission wavelength of 531 nm. The plate is equipped with shaking at 600 rpm for 3 s between readings controlled by software (Figure 1).</li>
	<li>Add an additional 4 &#956;L aliquot of 20 mM CaCl2 solution to each well and monitor the fluorescence changes every 3 s for 2 min. Repeat this step until the fluorescence increases with time. 
	NOTE: Mitochondria are challenged with the added Ca2+ indicated by the increased recording fluorescence. When the mPTP opens, the fluorescence increases with time.</li>
	<li>Interpret the total amount of Ca2+ added until the mPTP opens as the Ca2+ retention capacity.</li>
</ol>
3. Determination of Ca 2+-induced Mitochondrial Swelling
<ol>
	<li>Prepare the KCl media before the experiment.
	<ol>
		<li>Transfer 1 mL of isolated mitochondria (0.4 mg/mL) in KCl media into each 6-well plate well.</li>
	</ol>
	</li>
	<li>Add 10 &#956;L of 20 mM CaCl2 aqueous solution (500 nmol/mg mitochondrial protein) into mitochondrial protein to trigger mitochondrial swelling.</li>
	<li>Record the decrease in absorbance at 540 nm every 3 s on a microplate reader controlled by software for 10 min (Figure 2). The fluorescence changes are caused by an influx of solutes across the mitochondrial inner membrane. 
	NOTE: The decreased absorbance at 540 nm corresponds to the swollen mitochondria. If the reader does not observe a decrease in absorbance, a longer reading interval or reading time can be adopted.</li>
</ol>

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X. Sun supervised the experiments. Wei Li performed the experiments. Chen Zhang and X. Sun wrote the paper.

Here, we described a simple and effective protocol for the mitochondrial Ca2+ retention capacity assay and Ca2+-triggered mitochondrial swelling assay.
For the mitochondrial isolation, make sure that all the materials and tubes are on ice, especially when homogenizing the cells. 0.25% trypsin-EDTA can also be used to detach cells from the dish for 5 min at 37 &#176;C. After 15 - 25 strokes, it is necessary to observe the intact cell membrane under the microscope and avoid...

Mitochondria are the main cellular organs to produce nearly 95% of the ATP used in the mammalian cells by oxidative phosphorylation. It is reported that the sequestered micromolar concentration of Ca2+ by mitochondria, the presence of ADP, and inorganic phosphate can be used to phosphorylate ADP to synthesis ATP1. When the concentration of cytosolic Ca2+ goes above a threshold, mitochondria can uptake Ca2+ rapidly and efflux it slowly. Thus, the functioning mitochondria can influx the increased cytosolic Ca2+. Irrelevant to the participation in the oxidative phosphorylation, the mitochondrial Ca2+ also take part in the cytosolic calcium signals and activate the mitochondrial apoptotic mechanism by inducing the opening of the mPTP in the inner mitochondrial membrane2. It has been widely recognized that abnormal elevation of intracellular Ca2+ can induce mitochondrial massive swelling by opening the mPTP3. Thus, this protocol aims to assess the mitochondrial function by quantifying the mitochondrial Ca2+ retention capacity and Ca2+-trigged mitochondrial swelling.
Ca2+ retention capacity is the measurement of the ability of mitochondria to uptake cytosolic calcium. Mitochondria can buffer the cytosolic free calcium and regulate calcium-dependent cellular processes by calcium uptake in the form of inactive precipitates. Impaired Ca2+ retention capacity occurs in stress phenomena associated with energy limitation and even neurodegenerative diseases4,5. Isolated mitochondria or digitonin-permeabilized cells can be used to identify the Ca2+ retention capacity, and the elevated ability to accumulate Ca2+ by isolated mitochondria with the additonal glutamate and malate is not effected by the mitochondrial isolation procedure6. A titrated amount of digitonin should be used to permeabilize the plasma membranes of different cell types. The hexapotassium salt of Ca2+-binding green fluorescent dye, a Ca2+-sensitive cell-impermeant visible light-excitable indicator, has been widely used7. Ca2+-binding green fluorescent dye is a low-affinity indicator and used to show that intracellular free Ca2+ concentrations continue to rise during prolonged (5 min) stimulations8. This method was less sensitive than the radioactive filter technique but was greatly simplified. The additional Ca2+ elevates calcium green fluorescence and the Ca2+ uptake by mitochondria returns the fluorescence to baseline. Sequential additions of Ca2+ were made until the mitochondria failed to uptake extramitochondrial Ca2+ 9. A fluorescence microplate reader can be used to continuously report the calcium green fluorescence.
After Ca2+ accumulation, mitochondria depolarized, released Ca2+ into the medium, and began to swell. Ca2+-triggered mitochondria swelling is used to indicate the mPTP opening. Electron microscopy and the decrease in light absorbance at 540 nm can be used to measure the Ca2+-triggered mitochondrial swelling10,11. Mitochondria volume can be directly determined by forward angle light scattering12, where decreases in the absorbance reflect passive swelling of the mitochondrial matrix.
Here, we illustrate the methodology to examine the mitochondrial Ca2+ retention capacity and Ca2+-triggered mitochondrial swelling in isolated mitochondria from SH-SY5Y cells.

<table><tbody><tr><td>SH-SY5Y cell line</td><td>ATCC (The Global Bioresource Center)</td><td>ATCC Number: CRL-2266</td><td></td></tr><tr><td>Calcium green-5N</td><td>Life Technologies</td><td>c-3737</td><td>Ca&lt;sup&gt;2+&lt;/sup&gt;-binding green fluorescent dye</td></tr><tr><td>complete protease inhibitors</td><td>Roche Molecular Biochemicals</td><td>4693116001</td><td></td></tr><tr><td>glass homogenizer</td><td>Kimble Chase</td><td>9885303002</td><td></td></tr><tr><td>glutamate</td><td>Sigma-Aldrich</td><td>RES5063G-A7</td><td></td></tr><tr><td>malate</td><td>Sigma-Aldrich</td><td>46940-U</td><td></td></tr><tr><td>rotenone</td><td>Sigma-Aldrich</td><td>R8875</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>00457</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>V900050</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9541</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>V900266</td><td></td></tr><tr><td>Varioskan flash instruments</td><td>Thermo Scientific</td><td>IC100E</td><td></td></tr><tr><td>Bongkrekate</td><td>Biovision</td><td>1820-100</td><td></td></tr><tr><td>atractyloside</td><td>Sigma-Aldrich</td><td>C4992</td><td></td></tr><tr><td>high-glucose Dulbecco&amp;rsquo;s modified Eagle medium</td><td>Hyclone</td><td>SH30022</td><td>4.5 g/L glucose, L-glutamine, without sodium pyruvate</td></tr><tr><td>fetal bovine serum</td><td>Hyclone</td><td>SV30087</td><td></td></tr><tr><td>penicillin</td><td>Sigma-Aldrich</td><td>P3032</td><td></td></tr><tr><td>streptomycin</td><td>Invitrogen</td><td>11860-038</td><td></td></tr><tr><td>BCA assay kit</td><td>Thermo Scientific</td><td>NCI3225CH</td><td></td></tr><tr><td>PBS</td><td>Hyclone</td><td>SH30256.01</td><td></td></tr><tr><td>10 cm cell culture dish</td><td>NEST</td><td>704001</td><td></td></tr></tbody></table>

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.

Representative Results of Ca2+ Retention Capacity: 
Results were expressed as fluorescence values. With the additional pulses of Ca2+ (200 nmols/mg mitochondrial protein), the fluorescence increased by double above baseline with the mPTP opening. 5 &#956;M Bongkrekate (BKA), an inhibitor of Ca2+-induced mPTP opening, or 1 &#956;M atractyloside (ATR), an activator of Ca2+-induced mPTP opening, were added in isolated mitochondria. Th...

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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.

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 ...

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11.1K Views.  Qilu Hospital of Shandong University. 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.

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

Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy cells1-3. One approach for monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication4. We developed a sensitive technique to label newly synthesized mtDNA in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of 5-ethynyl-2'-deoxyuridine (EdU)5-7 with a tyramide signal amplification (TSA)8 protocol to visualize mtDNA replication within subcellular compartments of neurons. EdU is superior to other thymidine analogs, such as 5-bromo-2-deoxyuridine (BrdU), because the initial click reaction to label EdU5-7 does not require the harsh acid treatments or enzyme digests that are required for exposing the BrdU epitope. The milder labeling of EdU allows for direct comparison of its incorporation with other cellular markers9-10. The ability to visualize and quantify mtDNA biogenesis provides an essential tool for investigating the mechanisms used to regulate mitochondrial biogenesis and would provide insight into the pathogenesis associated with drug toxicity, aging, cancer and neurodegenerative diseases. Our technique is applicable to sensory neurons as well as other cell types. The use of this technique to measure mtDNA biogenesis has significant implications in furthering the understanding of both normal cellular physiology as well as impaired disease states.

We developed a sensitive technique to label newly synthesized mitochondrial DNA (mtDNA) in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of EdU together with a tyramide signal amplification (TSA) protocol to visualize mtDNA replication within subcellular compartments of neurons.

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy ...

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

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.

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 ...

Biochemistry

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

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 ...

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.

Test napływu wapnia w celu pomiaru mitochondrialnego wychwytu wapnia w hodowanych komórkach

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

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

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