This work was supported by HL094536 (B.J.H.).

1. Isolation of Mitochondria from Mouse Heart
<ol>
 <li>To isolate heart mitochondria, anesthetize and sacrifice mice according to the procedures approved by your local Institutional Animal Care and Use Committee.</li>
</ol>
Note: All steps of the mitochondria isolation protocol must be performed on ice. Use ice cold buffers and pre-chilled Petri dishes, Falcon tubes and Eppendorf tubes. The volumes given in the protocol are for a sample containing 2 mouse hearts.
<ol start="2">
 <li>Remove hearts, place them in cold Mitochondria Isolation Buffer (300 mM sucrose, 10 mM Na-HEPES, 200 &mu;M EDTA, pH 7.4) and rinse off blood.</li>
</ol>
Note: For optimal results, the pH of mitochondrial buffers should be adjusted with KOH and Acetic Acid.
<ol start="3">
 <li>Place hearts on a cold Petri dish, remove fat and atria, and mince finely using a blade until obtaining a homogeneous product.</li>
 <li>Transfer minced hearts to a 50 ml Falcon tube, add 10 ml of Mitochondria Isolation Buffer with 0.1 mg/ml trypsin and allow the sample to digest on ice for 10 min.</li>
</ol>
Note: Trypsin during the isolation procedure increases the yield of isolated mitochondria and should be kept constant between mitochondrial preparations. We recommend testing mitochondrial functions on preparations isolated is the absence of trypsin to ensure that the enzyme does not interfere with functionality. 
<ol start="5">
 <li>Add 10 ml of Mitochondria Isolation Buffer with 0.1% fatty acid free BSA and 5 mg trypsin inhibitor (soybean) and mix by inverting the tube several times to neutralize the trypsin.</li>
</ol>
Note: Mitochondria Isolation Buffer can be prepared in advance and can be stored at -20 &deg;C. Check pH upon thawing. Trypsin, trypsin inhibitor or BSA must be added to the Mitochondria Isolation Buffer on the day of the experiment.
<ol start="6">
 <li>Recover the digested tissue by low-to-medium speed centrifugation for 1 min at 4 &deg;C.</li>
 <li>Resuspend tissue in 8 ml Mitochondria Isolation Buffer with 0.1% fatty acid free BSA and homogenize using a motorized Dounce homogenizer with a Teflon pestle (4-6 passes at 1,200-14,00 rpm). Keep the sample on ice while homogenizing.</li>
 <li>Transfer homogenate into a 15 ml Falcon tube and centrifuge at 800xg for 10 min at 4 &deg;C to pellet nuclei and tissue debris.</li>
 <li>Recover the supernatant containing the mitochondria into Eppendorf tubes and centrifuge at 8,000 x g for 15 min, at 4 &deg;C.</li>
 <li>Remove carefully the top white layer and wash the remaining darker pellet containing the mitochondria in 1 ml Mitochondria Isolation Buffer.</li>
 <li>Centrifuge at 8000xg for 15 min at 4 &deg;C. Discard supernatant and resuspend mitochondria pellet in 150 &mu;l Mitochondrial Isolation Buffer. Keep mitochondria on ice for further assays.</li>
</ol>
Note: For best results during functional assays, use mitochondria within 4 hr of isolation.
<ol start="12">
 <li>Quantify mitochondrial protein using the Bradford assay.</li>
</ol>
2. Quality Control of Isolated Mitochondria
2.1. Measurement of respiratory control ratio (RCR)
<ol>
 <li>Calibrate the Oxytherm oxygen electrode with air-saturated water to establish the maximum oxygen line. When a stable level of oxygen is obtained add 100 &mu;l of a freshly prepared 10 mM sodium hydrosulfite solution to establish the zero oxygen line. Perform measurements at 30 &deg;C.</li>
 <li>Add 2 ml Mitochondria Respiration Buffer (125 mM KCl, 20 mM HEPES, 3 mM MgCl2, 400 &mu;M EGTA, 300 &mu;M DTT, 5 mM KH2PO4, pH 7.2) with 1 &mu;M rotenone to the Oxytherm chamber, seal the chamber and start recording.</li>
 <li>When oxygen signal is stable, add 250 &mu;g mitochondria and record basal respiration for 1 min. The respiration at this point should be very low as mitochondria are respiring on endogenous substrates (State 1 respiration).</li>
 <li>Add 5 mM succinate and record signal for 1 min. Oxygen consumption should increase during State 2 respiration.</li>
 <li>Induce State 3 respiration by adding 150 &mu;M ADP. At this point oxygen consumption should increase due to ATP synthesis through oxidative phosphorylation. Record signal until respiration slows, indicating ADP consumption and transition to State 4 respiration. Record State 4 respiration for at least 1 min.</li>
 <li>Add 1 &mu;M CCCP and record signal for 1 min. Uncoupled respiration should be maximal.</li>
</ol>
Note: The effect of CCCP on mitochondrial respiration is dose dependent: high concentrations of CCCP can inhibit oxygen consumption and should therefore be carefully titrated.
<ol start="7">
 <li>Calculate the respiratory control ratio (RCR) by dividing oxygen consumption rate during State 3 respiration by the oxygen consumption rate during State 4 respiration. A target RCR should be &ge; 4.</li>
</ol>
2.2. Assessment of mitochondrial membrane integrity (cytochrome c test)
<ol>
 <li>Thoroughly wash the Oxytherm chamber with 70% ethanol and water and repeat steps 2.1.2 -2.1.4.</li>
 <li>Add 1 mM ADP and record oxygen consumption for 1 min.</li>
 <li>Add 10 &mu;M cytochrome c. Since cytochrome c is impermeable to intact mitochondrial membranes, an increase in respiration indicates broken or damaged mitochondrial membranes.</li>
</ol>
3. Measurement of the Mitochondrial Permeability Transition Pore Opening
<ol>
 <li>Setup the spectrofluorometer for the multi-parameter measurement of mtPTP opening. Using the FelixGx program, create a new hardware configuration (Quanta master steady state) comprising the light source, excitation monochromator, sample compartment and two detectors positioned at 90 &deg; and 270 &deg; with respect to the excitation monochromator (Figure 4). To achieve maximal time resolution, both emission monochromators (90 &deg; and 270 &deg;) must be disabled and the emission filters at 90 &deg; and 270 &deg; in the sample compartment enabled in the hardware configuration menu. This step will remove the motor control of the emission wavelengths and fix each monochromator at a particular wavelength. If the emission monochromators are not disabled, each detector will cycle between both wavelengths and duplicate measurements will be recorded.</li>
 <li>Create a new acquisition protocol using the Multi Dye type and enter the following dyes:
 <ul>
 <li>Fura (high Ca++): excitation 340 nm, emission 525 nm (detector 1)</li>
 <li>Fura (low Ca++): excitation 380 nm, emission 525 nm (detector 1)</li>
 <li>JC1 (aggregate): excitation 543 nm, emission 595 nm (detector 2)</li>
 <li>JC1 (monomer): excitation 498 nm, emission 525 nm (detector 1)</li>
 <li>Swelling: excitation 525 nm, emission 525 nm (detector 1)</li>
 </ul>
 </li>
 <li>Set the excitation and emission slits to 1 nm and the temperature to 37 &deg;C.</li>
 <li>To obtain the ratiometric signal for Fura and JC-1, generate two derived traces: Fura (high Ca++)/Fura (low Ca++) and JC1 (aggregate)/JC1 (monomer).</li>
 <li>Manually adjust the emission wavelength of detectors 1 and 2 to 525 nm and 595 nm, respectively.</li>
 <li>Mix 1 ml Mitochondria Assay Buffer (120 mM KCl, 10 mM NaCl, 1 mM KH2PO4, 20 mM HEPES-Tris, pH 7.2) with 1 &mu;M rotenone, 5 mM succinate and 800 nM Fura FF in a disposable 4-sided methacrylate cuvette. Add 250 &mu;g mitochondria and place sample in the sample compartment. Make sure that the magnetic stirrer is turned on.</li>
 <li>Start recording. After 1 min, pause the acquisition, add 500 nM JC-1 and then restart the acquisition. The JC-1 ratio signal should increase, indicating dye uptake by active mitochondria. Continue recording until JC-1 signal has reached a plateau (usually within 5 min).</li>
 <li>Add 20 &mu;M CaCl2. An instantaneous increase in Fura FF ratio signal should be observed by the increased Ca2+ presence in the assay buffer, followed by a gradual decrease due to mitochondrial Ca2+ uptake. The JC-1 ratio signal should exhibit a brief transient decrease indicative of slight mitochondrial membrane depolarization.</li>
 <li>Wait until Fura FF ratio signal returns to basal level prior to addition of another CaCl2 pulse (1-1.5 min). Continue pulsing at fixed intervals until mitochondria cannot accumulate Ca2+ and begin releasing Ca2+ into the assay buffer. mtPTP opening is visualized by a concurrent increase in the Fura FF ratio signal due to Ca2+ release, a decrease in the JC-1 signal ratio due to membrane potential collapse, and a decrease in light scattering due to mitochondrial swelling (Figure 5).</li>
 <li>Following mtPTP activation, verify the specificity of Fura FF, JC-1 and swelling signals respectively with 1 mM EGTA, 1 &mu;M CCCP and 5 &mu;g/ml alamethicin.</li>
 <li>To verify specificity of mtPTP opening, repeat steps 3.6 - 3.9 in the presence of 1 &mu;M of the cyclophilin D inhibitor cyclosporine A (cyclophilin D is a component of the mtPTP). In the presence of cyclosporine A, opening of the mtPTP requires significantly more CaCl2 pulses than control samples (Figure 6).</li>
</ol>
4. Representative Results
Figure 2 shows a typical respiratory control of isolated mouse heart mitochondria. State 3 respiration is achieved by addition of ADP to mitochondria respiring on succinate, and is characterized by significantly increased oxygen consumption with respect to the substrate alone. Depletion of added ADP initiates State 4 respiration, during which oxygen consumption slows and is comparable to rates attained prior to ADP addition. RCR is obtained by dividing the oxygen consumption rate for State 3 respiration by that of State 4 respiration. The cytochrome c test is used to assay the integrity of the outer mitochondrial membrane: when cytochrome c is added to mitochondria respiring on succinate and ADP, no further increase in respiration is observed, indicating an intact outer mitochondrial membranes (Figure 3).
A representative image for the mtPTP opening in isolated heart mitochondria is shown in Figure 4. In this case mtPTP opening was triggered by the addition of 4 CaCl2 pulses (20 &mu;M each) at 1.5 min intervals. mtPTP opening is apparent by the near simultaneous release of mitochondrial Ca2+ (increase in Fura FF ratio signal), collapse of mitochondrial membrane potential (decrease in JC-1 ratio signal) and increased mitochondrial volume (decrease in the swelling signal). When cyclosporine A is added, which binds to the cyclophilin D component of mtPTP, mtPTP opening requires 7 pulses of CaCl2 instead of 4 (Figure 5).
<img alt="Figure 1" src="/files/ftp_upload/4131/4131fig1.jpg" /> 
Figure 1. Flow chart of the multi-parameter mitochondrial permeability transition pore opening protocol for isolated heart mitochondria. Heart is harvested from mice and mitochondria are isolated through differential centrifugation. The mitochondrial preparation is then qualitatively evaluated by polarographic measurement of the respiratory control ratio and mitochondrial membrane intactness in the presence of cytochrome c. Mitochondrial permeability transition pore opening in isolated mitochondria is triggered by sequential CaCl2 additions and is measured with a spectrofluorometer by monitoring Ca2+ release from mitochondria, membrane potential collapse and swelling of the mitochondrial matrix.
<img alt="Figure 2" src="/files/ftp_upload/4131/4131fig2.jpg" /> 
Figure 2. Measurement of respiratory control ratio (RCR) of isolated heart mitochondria. Oxygen consumption by isolated heart mitochondria is measured during State 3 respiration in the presence of succinate and ADP, and during State 4 respiration after ADP consumption. The response of isolated mitochondria to uncouplers is measured by the addition of CCCP. Numbers on the graph are the rates of oxygen consumption by isolated mitochondria in nmol/min/mg protein.
<img alt="Figure 3" src="/files/ftp_upload/4131/4131fig3.jpg" /> 
Figure 3. Measurement of membrane integrity of isolated heart mitochondria. Mitochondrial membrane integrity is determined by measuring oxygen consumption in isolated mitochondria in the presence of succinate and ADP and after the addition of cytochrome c. Numbers on the graph are the rates of oxygen consumption by isolated mitochondria in nmol/min/mg protein.
<img alt="Figure 4" src="/files/ftp_upload/4131/4131fig4.jpg" /> 
Figure 4. Spectrofluorometer hardware configuration for the multi-parameter measurement of mtPTP opening. The hardware configuration of the spectrofluorometer includes a light source, an excitation monochromator, a sample compartment and two detectors with fixed emission wavelengths at 525 nm and 595 nm. Detector 1 is used for the signal detection of Fura FF high and low Ca2+, JC-1 monomer, and the swelling signal. Detector 2 measures the JC-1 aggregate signal.
<img alt="Figure 5" src="/files/ftp_upload/4131/4131fig5.jpg" /> 
Figure 5. Multi-parameter measurement of mtPTP opening in isolated heart mitochondria. mtPTP opening was triggered by sequential additions of 20 &mu;M CaCl2 and was characterized by Ca2+ release from mitochondria, mitochondrial membrane potential collapse and increased mitochondrial matrix volume (swelling). Extramitochondrial Ca2+ and membrane potential were measured with the ratiometric indicators Fura FF (green trace) and JC-1 (red trace). Swelling of mitochondria was measured by recording light scattering at 525 nm (black trace). Specificity of JC-1 and swelling signals was tested with 1 &mu;M CCCP and 5 &mu;g/ml alamethicin (a microbial toxin).
<img alt="Figure 6" src="/files/ftp_upload/4131/4131fig6.jpg" /> 
Figure 6. Multi-parameter measurement of mtPTP opening in isolated heart mitochondria in the presence of cyclosporine A. Cyclosporine A (1 &mu;M) increases the number of CaCl2 pulses required to open mtPTP in isolated heart mitochondria.

<ol>
	<li>Kroemer, G., Galluzzi, L., Brenner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Membrane+Permeabilization+in+Cell+Death.">Mitochondrial Membrane Permeabilization in Cell Death.</a> Physiol. Rev. 87, 99-163 (2007).</li><li>Elrod, J., Wong, R., Mishra, S., Vagnozzi, R. J., Sakthievel, B., Goonasekera, S. A., Karch, J., Gabel, S., Farber, J., Force, T., Brown, J. H., Murphy, E., Molkentin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclophilin+D+controls+mitochondrial+pore-dependent+Ca2++exchange,+metabolic+flexibility,+and+propensity+for+heart+failure+in+mice.">Cyclophilin D controls mitochondrial pore-dependent Ca2+ exchange, metabolic flexibility, and propensity for heart failure in mice.</a> J. Clin. Invest. 120, 3680-3687 (2010).</li><li>Hom, J. R., Quintanilla, R. 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No conflicts of interest declared.

The protocol presented here describes the necessary experimental steps to assess permeability transition pore opening in isolated heart mitochondria (Figure 1 and Figure 4): the procedure for isolating mouse heart mitochondria, the respiratory controls that ensure their integrity and functionality, the mitochondrial parameters monitored during mtPTP opening and the dyes employed for their measurement, the setting up of the spectrofluorometric instrumentation, and the characterization of ...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T3030</td>
		</tr>
		<tr>
			<td>Trypsin inhibitor (soybean)</td>
			<td>Sigma-Aldrich</td>
			<td>T9128</td>
		</tr>
		<tr>
			<td>Sodium hydrosulfite</td>
			<td>Sigma-Aldrich</td>
			<td>71699</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma-Aldrich</td>
			<td>R8875</td>
		</tr>
		<tr>
			<td>Cytochrome c</td>
			<td>Sigma-Aldrich</td>
			<td>C7752</td>
		</tr>
		<tr>
			<td>Alamethicin</td>
			<td>Sigma-Aldrich</td>
			<td>A4665</td>
		</tr>
		<tr>
			<td>CCCP</td>
			<td>Sigma-Aldrich</td>
			<td>C2759</td>
		</tr>
		<tr>
			<td>Cyclosporin A</td>
			<td>Calbiochem</td>
			<td>239835</td>
		</tr>
		<tr>
			<td>Fura FF</td>
			<td>Invitrogen</td>
			<td>F14180</td>
		</tr>
		<tr>
			<td>JC-1</td>
			<td>Invitrogen</td>
			<td>T3168</td>
		</tr>
		<tr>
			<td>Tissue grinder Potter-Elvehjem with Teflon pestle 15 ml</td>
			<td>Wheaton Industries</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Overhead stirrer</td>
			<td>Wheaton Industries</td>
			<td>903475</td>
		</tr>
		<tr>
			<td>Oxytherm (temperature controlled oxygen electrode)</td>
			<td>Hansatech Instruments</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>QuantaMaster 80 dual emission spectrofluorometer</td>
			<td>Photon Technology International, Inc.</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria at JoVE.com

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

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21.4K visualizações.  University of Washington, Seattle. Um protocolo espectrofluorométrico para a medição da abertura do poro de permeabilidade mitocondrial transição de coração de rato isolado mitocôndrias é aqui apresentada. O teste envolve a medição simultânea de Ca mitocôndrias 2 + Manuseamento, volume potencial de membrana mitocondrial e mitocondrial. O procedimento para a obtenção de coração e de alta qualidade funcional a mitocôndria é também descrito.

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

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of myocardial metabolism have been linked to the development of contractile dysfunction and heart failure. Therefore, unraveling the link between metabolism and contraction should shed light on some of the mechanisms governing cardiac adaptation or maladaptation in disease states. The isolated working rat heart preparation can be used to follow, simultaneously and in real time, cardiac contractile function and flux of energy providing substrates into oxidative metabolic pathways. The present protocol aims to provide a detailed description of the methods used in the preparation and utilization of buffers for the quantitative measurement of the rates of oxidation for glucose and fatty acids, the main energy providing substrates of the heart. The methods used for sample analysis and data interpretation are also discussed. In brief, the technique is based on the supply of 14C- radiolabeled glucose and a 3H- radiolabeled long-chain fatty acid to an ex vivo beating heart via normothermic crystalloid perfusion. 14CO2 and 3H2O, end byproducts of the enzymatic reactions involved in the utilization of these energy providing substrates, are then quantitatively recovered from the coronary effluent. With knowledge of the specific activity of the radiolabeled substrates used, it is then possible to individually quantitate the flux of glucose and fatty acid in the oxidation pathways. Contractile function of the isolated heart can be determined in parallel with the appropriate recording equipment and directly correlated to metabolic flux values. The technique is extremely useful to study the metabolism/contraction relationship in response to various stress conditions such as alterations in pre and after load and ischemia, a drug or a circulating factor, or following the alteration in the expression of a gene product.

The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found responsible for very diverse diseases, ranging from neurological pathologies to cancer. Interestingly, mitochondria have recently been shown to display the capacity to be transferred between cell types, notably from human mesenchymal stem cells (MSC) to cancer cells in coculture conditions, with metabolic and functional consequences for the mitochondria recipient cells, further enhancing the current interest for the biological properties of these organelles.
Evaluating the effects of the transferred MSC mitochondria in the target cells is of primary importance to understand the biological outcome of such cell-cell interactions. The MitoCeption protocol described here allows the transfer of the mitochondria isolated beforehand from the donor cells to the target cells, using MSC mitochondria and glioblastoma stem cells (GSC) as a model system. This protocol has previously been used to transfer mitochondria, isolated from MSCs, to adherent MDA-MB-231 cancer cells. This mitochondria transfer protocol is adapted here for GSCs that present the specific particularity of growing as neurospheres in vitro. The transfer of the isolated mitochondria can be followed by fluorescence-activated cell sorting (FACS) and confocal imaging using mitochondria vital dyes. The use of mitochondria donor and target cells with distinct haplotypes (SNPs) also allows detection of the transferred mitochondria based on the concentration of their circular mitochondrial DNA (mtDNA) in the target cells. Once the protocol has been validated with these criteria, the cells harboring the transferred mitochondria can be further analyzed to determine the effects of the exogenous mitochondria on biological properties such as cell metabolism, plasticity, proliferation and response to therapy.

Here, a protocol (MitoCeption) is presented to transfer mitochondria, isolated from human mesenchymal stem cells (MSC), to glioblastoma stem cells (GSC), with the goal of studying their biological effects on GSC metabolism and functions. A similar protocol can be adapted to transfer mitochondria between other cell types.