Name: isolation of intact mitochondria from skeletal muscle by differential centrifugation for high-resolution respirometry measurements
Uploaded: 2017-03-08T14:00:00.000+00:00
Duration: 10 min 25 s
Description: Watch this Scientific Journal Video about Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements at JoVE.com

This study was supported by the Swiss National Science Foundation (Grant 32003B_127619).

The quadriceps muscle biopsy is taken from an anaesthetized pig, from which mitochondria are isolated by differential centrifugation. The pig is used afterwards for another experiment. The study is performed in accordance with the National Institutes of Health guidelines for the care and use of experimental animals and with the approval of the Animal Care Committee of the Canton Bern, Switzerland.
1. Skeletal Muscle Homogenization and Mitochondrial Isolation
<ol>
	<li>Excise 5-10 g quadriceps muscle specimen from an anaesthetized pig. In the present assay, use porcine skeletal muscle. This protocol can also be used to isolate mitochondria from other species (e.g., rat, mice, human, etc.). 
	NOTE: This step is performed by a medical specialist with expertise in surgery.
	<ol>
		<li>Sedate pigs with intramuscular ketamine (20 mg/kg) and xylazine (2 mg/kg), before anesthesia is induced with intravenous midazolam (0.5 mg/kg, plus atropine 0.02 mg/kg). Maintain anesthesia with continuous intravenous infusions of propofol (4-8 mg/kg per h) and fentanyl (30 &#181;g/kg per h) during surgery.</li>
	</ol>
	</li>
	<li>Immediately immerse the tissue sample in a beaker containing 20 mL of ice-cold mitochondrial isolation buffer (Table 1). 
	NOTE: Buffers used during homogenization and centrifugations should be ice cold and have a physiological relevant pH with an ionic and osmotic strength compatible with cytosol.</li>
	<li>Weigh the tissue sample in the beaker on an analytical tared balance. Tare the balance with the beaker containing 20 mL of isolation buffer.</li>
	<li>Place the beaker on an ice bucket. 
	NOTE: Perform all of the next steps for the homogenization procedure at the ice bucket temperature and keep all buffers on the ice bucket.</li>
	<li>Mince the skeletal muscle in the beaker (keep on ice) into 1-2 mm small pieces for 3-4 min using fine scissors.</li>
	<li>Rinse the minced tissue in the beaker twice with ice cold isolation buffer (20 mL each washing step).</li>
	<li>Suspend the tissue in 10 volumes of the isolation buffer per tissue weight (g) containing 5 mg protease/g tissue.</li>
	<li>Place the beaker in an ice bath on the magnetic stirrer and stir for 10 min.</li>
	<li>Dilute the suspension in the beaker with additional 10 volumes of the isolation buffer per tissue weight (g) supplemented with 0.2% (weight/volume) defatted bovine serum albumin (BSA).</li>
	<li>Decant all of the isolation buffer supplemented with 0.2% BSA and rinse the tissue in the beaker twice with ice cold isolation buffer supplemented with 0.2% BSA (20 mL each washing step).</li>
	<li>Resuspend the tissue in 10 volumes of isolation buffer per tissue weight (g) supplemented with 0.2% with defatted BSA.</li>
	<li>Homogenize the tissue with a semi-automatic glass homogenizer (kept on ice) with a loose-fitting pestle (10 strokes). 
	NOTE: Homogenize the tissue always in the same manner (the same number of strokes, e.g., 10 strokes). A higher number of strokes may damage and uncouple the mitochondria. Preferably, homogenize the tissue using an automated homogenizer. Manually (and subjectively) homogenized tissue samples in glass homogenizers may produce different qualities of isolated mitochondria.</li>
	<li>Centrifuge the homogenized tissue at 4 &#176;C for 10 min at 10,000 x g. 
	NOTE: All centrifugation steps are performed in centrifuges with fixed angle rotors.</li>
	<li>Discard the supernatant using a serological pipet and resuspend the pellet in BSA-supplemented ice-cold isolation medium (10 mL/g tissue).</li>
	<li>Centrifuge the suspension at 4 &#176;C for 10 min at 350 x g.</li>
	<li>Reserve the supernatant using a serological pipet and discard the pellet (cellular debris).</li>
	<li>Filter the supernatant into a beaker (kept on ice) through two layers of gauze (17 threads/cm2) to remove cell debris.</li>
	<li>Centrifuge the filtered suspension at 4 &#176;C for 10 min at 7,000 x g.</li>
	<li>Discard the supernatant and resuspend the crude mitochondrial pellet in the isolation buffer (5 mL/g tissue) supplemented with 0.2% (weight/volume) BSA and centrifuge the suspension at 4 &#176;C for 10 min at 7,000 x g.</li>
	<li>Discard the supernatant and resuspend the crude mitochondrial pellet in wash buffer (Table 1). Centrifuge the suspension again at 4 &#176;C for 10 min at 7,000 x g.</li>
	<li>Discard the supernatant and resuspend the crude mitochondrial pellet in wash buffer and centrifuge the suspension again at 4 &#176;C for 10 min at 7,000 x g.</li>
	<li>Discard the supernatant and resuspend the crude mitochondrial pellet in 1.0 mL of wash buffer.</li>
	<li>Determine the protein concentration of the mitochondrial suspension and keep the concentrated mitochondrial suspension on ice. 
	NOTE: Protein concentration can be measured using any standard method.</li>
	<li>Just prior to respirometry assays, resuspend the mitochondrial suspension in the respiration buffer to a final concentration of 0.4 mg/mL mitochondrial protein.</li>
</ol>
2. High-resolution Respirometry
<ol>
	<li>Pipette 2.1 mL of respiration buffer (Table 1) 16 into a high-resolution oxygraph chamber and stir the buffer continuously using a magnetic stirring bar present in the chamber (700 rpm) at 37 &#176;C for 1 h until a stable oxygen flux signal of the polarographic oxygen sensor is obtained. 
	NOTE: Polarographic oxygen electrodes within each oxygraph chamber measure the oxygen concentration and calculate oxygen consumption (flux) within each chamber. The oxygen concentration and oxygen consumption rates (flux) are displayed real-time online in a computer using software for data acquisition and analysis. In addition, in order to ensure accurate and reliable results, instrumental oxygen background correction should be performed according to the manufacturer&#39;s instructions. 
	NOTE: Accurately 2.1 mL of respiration buffer is added into the chamber for calibration of a final chamber volume of 2.0 mL after closing the stoppers.</li>
	<li>Perform an air calibration of the polarographic oxygen sensor according to the manufacturer&#39;s protocols 17. 
	NOTE: During air calibration, with a gas phase present, the media oxygen concentration will equilibrate on the order of 15-20 min. Depending on the temperature, barometric pressure, and solubility of media -oxygen concentration can be calculated.</li>
	<li>After air calibration, aspirate the respiration medium from the oxygraph chamber and add 2.1 mL of isolated mitochondrial suspension (0.4 mg/mL mitochondrial protein) from step 1.24 to a chamber of the oxygraph. 
	NOTE: The volume of respiration buffer depends on the instrument set up and manufacturer. For high resolution respirometry, the 2.1 mL of respiration buffer is added into the chamber for calibration of a final chamber volume of 2.0 mL after closing the stoppers.</li>
	<li>Close the oxygraph chamber by insertion of the stopper.</li>
	<li>Stir the mitochondrial suspension continuously using a magnetic stirring bar present in the chamber (700 rpm) at 37 &#176;C and record cellular respiration at baseline for 3-5 min until a stable oxygen flux signal is achieved. 
	NOTE: The oxygen concentration and oxygen flux signal are displayed real-time online in a computer using software for data acquisition and analysis 17.</li>
	<li>Afterwards, inject substrates and inhibitors for mitochondrial respiration through the titanium injection ports of the stoppers using the following standard protocols.</li>
</ol>
3. Complex I-dependent Respiration
<ol>
	<li>Prepare an oxygraph chamber containing the isolated mitochondrial suspension (0.4 mg/mL) following the procedure described in steps 2.1-2.5 and wait for a stable signal.</li>
	<li>Inject 12.5 &#181;L of 0.8 M malate (5 mM final concentration) and 10 &#181;L of 2 M glutamate (10 mM final concentration) into the oxygraph chamber containing isolated mitochondrial suspension (0.4 mg/mL) through the titanium injection port of the chamber stopper and record cellular respiration for 3-5 min until a stable oxygen flux signal is achieved. 
	NOTE: All the injections in the following steps are performed through the titanium injection ports of stoppers using syringes.</li>
	<li>Inject 10 &#181;L of 0.05 M ADP (0.25 mM) into the oxygraph chamber through the titanium injection port of the chamber stopper and record mitochondrial respiration until the oxygen flux signal increases, then decreases and stabilizes (3-5 min). 
	NOTE: The plateau of respiration after ADP addition indicates that ADP concentration was high and saturating. Depending on the amount of mitochondria used during respiration, ADP concentration should be titrated for a stable state 3 oxygen consumption (maximal respiration). Addition of ADP to the mitochondrial suspension will induce an increase in oxygen consumption and the oxygen flux signal will increase (state 3 respiration). After the ADP is consumed, the oxygen flux signal will decrease (state 4 respiration).</li>
</ol>
4. Complex II-dependent Respiration
<ol>
	<li>Prepare an oxygraph chamber containing isolated mitochondrial suspension (0.4 mg/mL) following the procedure described in steps 2.1-2.5 and wait for a stable signal.</li>
	<li>Inject 2 &#181;L of 0.2 mM rotenone (0.2 &#181;M) &#39;CAUTION&#39; into the oxygraph chamber through the titanium injection port of the chamber stopper using a syringe and record the cellular respiration for 3-5 min until a stable oxygen flux signal is achieved. 
	NOTE: Rotenone is a dangerous poison and may have a significant impact on human health.</li>
	<li>Inject 20 &#181;L of 1 M succinate (10 mM) into the oxygraph chamber and record mitochondrial respiration for 3-5 min until a stable oxygen flux signal is achieved.</li>
	<li>Inject 10 &#181;L of 0.05 M ADP (0.25 mM) into the oxygraph chamber through the titanium injection port of the chamber stopper and record cellular respiration until the oxygen flux signal increases, stabilizes, then decreases and stabilizes (3-5 min). 
	NOTE: Addition of ADP to the mitochondrial suspension will induce an increase in oxygen consumption and the oxygen flux signal will increase (state 3 respiration). After the ADP is consumed, the oxygen flux signal will decrease (state 4 respiration).</li>
</ol>
5. Complex IV-dependent Respiration
<ol>
	<li>Prepare an oxygraph chamber containing isolated mitochondrial suspension (0.4 mg/mL) following the procedure described in steps 2.1 to 2.5 of the protocol and wait for a stable signal.</li>
	<li>Then inject 2.5 &#181;L of 0.8 mM ascorbate (1 mM). Immediately thereafter inject 2.5 &#181;L of 0.2 M TMPD (0.25 mM) and 10 &#181;L of 0.05 M ADP (0.25 mM) into the oxygraph chamber and record cellular respiration until the oxygen flux signal increases and stabilizes (3-5 min).</li>
	<li>Finally inject 10 &#181;L of 1 M sodium azide (5 mM) &#39;CAUTION&#39; into the oxygraph chamber and record cellular respiration until the oxygen flux signal decreases and stabilizes. 
	NOTE: Sodium azide is a dangerous poison and may have a significant impact on human health.</li>
</ol>
6. Cytochrome C Test
<ol>
	<li>Prepare an oxygraph chamber containing isolated mitochondrial suspension (0.4 mg/mL) following the procedure described in steps 2.1 to 2.5 of the protocol and wait for a stable signal.</li>
	<li>Inject 12.5 &#181;L of 0.8 M malate (5 mM final concentration) and 10 &#181;L of 2 M glutamate (10 mM final concentration) into the oxygraph chamber containing isolated mitochondrial suspension (0.4 mg/mL) through the titanium injection port of the chamber stopper and record cellular respiration for 3-5 min until a stable oxygen flux signal is achieved.</li>
	<li>Inject 10 &#181;L of 0.5 M ADP (2.5 mM) into the oxygraph chamber through the titanium injection port of the chamber stopper and record mitochondrial respiration until the oxygen flux signal increases, then decreases and stabilizes (3-5 min).</li>
	<li>Finally, inject 5 &#181;L of 4 mM cytochrome c (10 &#181;M) into the oxygraph chamber and record cellular respiration for 5 min until a stable oxygen flux signal is achieved.</li>
</ol>

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The authors have nothing to disclose.

In the present study we describe a protocol to isolate high-quality, intact and tightly coupled skeletal muscle mitochondria by differential centrifugation which can be used for functional studies such as high-resolution respirometry.
In order to isolate intact and tightly coupled mitochondria, there are some critical points to be considered within the present protocol. After harvesting the skeletal tissue, it should be immediately immersed in ice-cold mitochondrial isolation buffer. All centr...

Mitochondrial bioenergetics and respiratory capacities can be studied not only in permeabilized cells or fibers but also in isolated mitochondria. In the present study, we describe a protocol to isolate intact skeletal muscle mitochondria using differential centrifugation for high-resolution respirometry measurements.
To isolate intact mitochondria for respirometry, the tissue is homogenized and mitochondria are isolated by a conventional differential centrifugation method. The differential centrifugation method is based on sequential centrifugations (in a series of increasing speed) of tissue homogenates was first introduced by Pallade and co-workers almost 70 years ago 1. The tissue is first minced using scissors and homogenized mechanically in a glass homogenizer with a loose-fitting pestle. Afterwards the homogenate is centrifuged at low speed and the resulting pellet that contains unbroken tissue, cellular debris and nuclei is discarded. Then, the supernatant is centrifuged several times at high speed and the mitochondrial enriched fraction is collected. The advantages of the differential centrifugation method to isolate mitochondria are that: i) the method is fast and mitochondria can be isolated within 1-1.5 h (respiratory experiments should be performed as quick as possible); ii) it is inexpensive; and iii) it is very efficient and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. The disadvantages of differential centrifugation method to isolate mitochondria are that i) mitochondria might get damaged and uncoupled during homogenization; ii) the contamination of mitochondria with other cellular components (could be solved by further washing the mitochondrial pellet with additional centrifugation steps); iii) the possibility of selecting different mitochondrial subpopulations, e.g., during differential centrifugations steps, mitochondria with lower dense can be excluded 7; and iv) the mitochondrial cellular surrounding is missing and only the theoretical maximal respiration can be measured. Another method to isolate mitochondria for respirometry assays is the density gradient centrifugation 2. In this technique, the tissue extract is layered over a solution of sucrose or a Percoll gradient (with higher density at the bottom of the centrifugation tube) and centrifuged at a certain speed, causing the mitochondria to be isolated from other cellular components according to their densities. This method is often used to isolate brain mitochondria with very low contamination from synaptosomes. However, the rat liver mitochondria isolated by density gradient centrifugation are highly contaminated with other cellular organelles 3. One of the limitations of this method is that the sucrose gradient present in the centrifugation tube might rupture some mitochondria (osmotic shock).
Depending on the type of tissue; there are some important factors to consider for the isolation of intact mitochondria by differential centrifugation. The first necessity is to homogenize tissues in a gentle manner. Soft tissues such as kidney, brain and liver require gentle mechanical forces applied during homogenization. This contrasts with hard tissues such as cardiac and skeletal muscle that require much stronger mechanical forces. The minced tissue is usually treated with proteinase prior to the homogenization to soften the tissue. All buffers used during homogenization and centrifugation should be ice cold and have a physiological relevant pH with an ionic and osmotic strength compatible with cytosol 4,5.
One of the advantages of studying isolated mitochondrial bioenergetics is that cellular plasma membranes do not need to be permeabilized with detergents such as digitonin or saponin 4,6, which might compromise the mitochondrial outer membrane integrity. Another advantage of the isolated mitochondria is the absence of other cytosolic factors, which may interfere with the analysis of the mitochondrial functions such as oxygen consumption. The disadvantages of using the isolated mitochondria are the possible selection of certain mitochondrial populations during the centrifugation steps, damage to the mitochondria during homogenization, and the requirement for high quantities of biological samples in order to obtain a good yield of isolated mitochondria 7,8.
After the isolation procedure, the respiratory rates of mitochondrial complexes I-, II- and IV-dependent (states 2, 3 and 4) are determined using high-resolution respirometry. For complex I-driven respiration, glutamate and malate are added followed by adenosine diphosphate (ADP). For complex II-driven respiration, succinate is added followed by ADP. For complex IV-driven respiration, ascorbate and tetramethylphenylendiamine (TMPD) are added followed by ADP 9,10,11,12. State 2 refers to oxygen consumption in the presence of substrates alone. State 3 refers to oxygen consumption in the presence of substrates and ADP. State 4 refers to oxygen consumption after ADP depletion. The respiratory control ratio (RCR) is an index of coupling of oxygen consumption ATP production and is calculated as the ratio between state 3 and state 4 13,15.
In summary, we describe a protocol to isolate functional and intact skeletal muscle mitochondria by differential centrifugation and use these isolated mitochondria for functional and bioenergetic studies such as high-resolution respirometry.

<table><tbody><tr><td>ADP</td><td>Sigma</td><td>A 4386</td><td>Chemical</td></tr><tr><td>Antimycin A</td><td>Sigma</td><td>A 8674</td><td>Chemical, dissolve in ethanol</td></tr><tr><td>Ascorbate</td><td>Merck</td><td>1.00127</td><td>Chemical</td></tr><tr><td>ATP</td><td>Sigma</td><td>A 7699</td><td>Chemical</td></tr><tr><td>BSA</td><td>Sigma</td><td>A 6003</td><td>Chemical</td></tr><tr><td>EGTA</td><td>fluka</td><td>3779</td><td>Chemical</td></tr><tr><td>Glutamate</td><td>Sigma,</td><td>G 1626</td><td>Chemical</td></tr><tr><td>Hepes</td><td>Sigma</td><td>H 7523</td><td>Chemical</td></tr><tr><td>KCl</td><td>Merck</td><td>1.04936</td><td>Chemical</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck</td><td>1.04873</td><td>Chemical</td></tr><tr><td>K-lactobionate</td><td>Sigma</td><td>L 2398</td><td>Chemical</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M 9272</td><td>Chemical</td></tr><tr><td>Morpholinopropane sulphonic acid (MOPS)</td><td>Merck</td><td>1.06129</td><td>Chemical</td></tr><tr><td>O2k-Core: Oxygraph-2k&amp;nbsp;</td><td>Oroboros Instruments</td><td>10000-02</td><td>High-resolution respirometry instrument</td></tr><tr><td>Proteinase, bacterial</td><td>Sigma</td><td>P 8038</td><td>Chemical</td></tr><tr><td>Sodium azide</td><td>Sigma</td><td>S2002</td><td>Chemical</td></tr><tr><td>Rotenone</td><td>Sigma</td><td>R 8875</td><td>Chemical, dissolve in ethanol</td></tr><tr><td>Succinate</td><td>Sigma</td><td>S 2378</td><td>Chemical</td></tr><tr><td>Schuett homogen-plus semiautomatic homogeniser&amp;nbsp;</td><td>schuett-biotec GmbH</td><td>3.201 011</td><td>Tissue homogenizer</td></tr><tr><td>Taurine</td><td>Sigma</td><td>T 8691</td><td>Chemical</td></tr><tr><td>TMPD</td><td>Sigma</td><td>T 3134</td><td>Chemical</td></tr></tbody></table>

isolation of intact mitochondria from skeletal muscle by differential centrifugation for high-resolution respirometry measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Complex I-dependent respiration
Isolated mitochondrial complex I-dependent respiratory rates (states 2, 3 and 4) are determined using high-resolution respirometry (Figure 1, a representative diagram). Mitochondrial complex I substrates, glutamate and malate, are added followed by the addition of ADP. State 2 refers to oxygen consumption in the presence of the substrates alone. State 3 refers to ...

Watch this Scientific Journal Video about Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements at JoVE.com

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

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Research

JoVE Journal

Biology

18.0K Views.  Inselspital, Bern University Hospital. Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

Video: Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, mitochondria exist in a series of complex morphologies, ranging from small oval organelles to a broad, reticulum-like network. Understanding how the mitochondrial reticulum expands and develops in response to diverse stimuli such as alterations in energy demand has long been a topic of research. A key aspect of this growth, or biogenesis, is the import of precursor proteins, originally encoded by the nuclear genome, synthesized in the cytosol, and translocated into various mitochondrial sub-compartments. Mitochondria have developed a sophisticated mechanism for this import process, involving many selective inner and outer membrane channels, known as the protein import machinery (PIM). Import into the mitochondrion is dependent on viable membrane potential and the availability of organelle-derived ATP through oxidative phosphorylation. Therefore its measurement can serve as a measure of organelle health. The PIM also exhibits a high level of adaptive plasticity in skeletal muscle that is tightly coupled to the energy status of the cell. For example, exercise training has been shown to increase import capacity, while muscle disuse reduces it, coincident with changes in markers of mitochondrial content. Although protein import is a critical step in the biogenesis and expansion of mitochondria, the process is not widely studied in skeletal muscle. Thus, this paper outlines how to use isolated and fully functional mitochondria from skeletal muscle to measure protein import capacity in order to promote a greater understanding of the methods involved and an appreciation of the importance of the pathway for organelle turnover in exercise, health, and disease.

Mitochondria are key metabolic organelles that exhibit a high level of phenotypic plasticity in skeletal muscle. The import of proteins from the cytosol is a critical pathway for organelle biogenesis, essential for the expansion of the reticulum and the maintenance of mitochondrial function. Therefore, protein import serves as a barometer of cellular health.

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, ...

Biochemistry

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Medicine

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Mitochondrial Isolation from Rodents for High-Quality Bioenergetic Studies

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry to study metabolic impairments and disease. Consistent mitochondrial quality is necessary to properly investigate mitochondrial physiology and bioenergetics; however, many different published isolation methods are available for researchers. Although different experimental strategies require different isolation methods, the basic principles and procedures are similar. This protocol details a method capable of extracting well-coupled mitochondria from a variety of tissue sources, including small animals and cells. The steps outlined include organ dissection, mitochondrial purification, protein quantification, and various quality control checks. The primary quality control metric used to identify high-quality mitochondria is the respiratory control ratio (RCR). The RCR is the ratio of the respiratory rate during oxidative phosphorylation to the rate in the absence of ADP. Alternative metrics are discussed. While high RCR values relative to their tissue source are obtained using this protocol, several steps can be optimized to suit the individual needs of researchers. This procedure is robust and has consistently resulted in isolated mitochondria with above-average RCR values across animal models and tissue sources.

Author Spotlight: Uncovering the Role of Mitochondrial Calcium Phosphate in Heart Failure and Bioenergetics

Bioenergetic and metabolomic studies on mitochondria have revealed their multifaceted role in many diseases, but the isolation methods for these organelles vary. The method detailed here is capable of purifying high-quality mitochondria from multiple tissue sources. Quality is determined by respiratory control ratios and other metrics assessed with high-resolution respirometry.

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

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 ...

Development of a Mobile Mitochondrial Physiology Laboratory for Measuring Mitochondrial Energetics in the Field

Mitochondrial energetics is a central theme in animal biochemistry and physiology, with researchers using mitochondrial respiration as a metric to investigate metabolic capability. To obtain the measures of mitochondrial respiration, fresh biological samples must be used, and the entire laboratory procedure must be completed within approximately 2 h. Furthermore, multiple pieces of specialized equipment are required to perform these laboratory assays. This creates a challenge for measuring mitochondrial respiration in the tissues of wild animals living far from physiology laboratories as live tissue cannot be preserved for very long after collection in the field. Moreover, transporting live animals over long distances induces stress, which can alter mitochondrial energetics.
This manuscript introduces the Auburn University (AU) MitoMobile, a mobile mitochondrial physiology laboratory that can be taken into the field and used on-site to measure mitochondrial metabolism in tissues collected from wild animals. The basic features of the mobile laboratory and the step-by-step methods for measuring isolated mitochondrial respiration rates are presented. Additionally, the data presented validate the success of outfitting the mobile mitochondrial physiology laboratory and making mitochondrial respiration measurements. The novelty of the mobile laboratory lies in the ability to drive to the field and perform mitochondrial measurements on the tissues of animals captured on site.

We designed and constructed a mobile laboratory to measure respiration rates in isolated mitochondria of wild animals captured at field locations. Here, we describe the design and outfitting of a mobile mitochondrial laboratory and the associated laboratory protocols.

Mitochondrial energetics is a central theme in animal biochemistry and physiology, with researchers using mitochondrial respiration as a metric to ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...

High-Resolution Fluoro-Respirometry of Equine Skeletal Muscle

Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring mitochondrial function is fundamental in biomedical research. Skeletal muscle is a robust source of mitochondria, particularly in animals with a very high aerobic capacity, such as horses, making them ideal subjects for studying mitochondrial physiology. This article demonstrates the use of high-resolution respirometry with concurrent fluorometry, with freshly harvested skeletal muscle mitochondria, to quantify the capacity to oxidize substrates under different mitochondrial states and determine the relative capacities of distinct elements of mitochondrial respiration. Tetramethylrhodamine methylester is used to demonstrate the production of mitochondrial membrane potential resulting from substrate oxidation, including calculation of the relative efficiency of the mitochondria by calculating the relative membrane potential generated per unit of concurrent oxygen flux. The conversion of ADP to ATP results in a change in the concentration of magnesium in the reaction chamber, due to differing affinities of the adenylates for magnesium. Therefore, magnesium green can be used to measure the rate of ATP synthesis, allowing the further calculation of the oxidative phosphorylation efficiency (ratio of phosphorylation to oxidation [P/O]). Finally, the use of Amplex UltraRed, which produces a fluorescent product (resorufin) when combined with hydrogen peroxide, allows the quantification of reactive oxygen species production during mitochondrial respiration, as well as the relationship between ROS production and concurrent respiration. These techniques allow the robust quantification of mitochondrial physiology under a variety of different simulated conditions, thus shedding light on the contribution of this critical cellular component to both health and disease.

Horses have an exceptional aerobic exercise capacity, making equine skeletal muscle an important tissue for both the study of equine exercise physiology as well as mammalian mitochondrial physiology. This article describes techniques for the comprehensive assessment of mitochondrial function in equine skeletal muscle.

Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring ...