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 q...

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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>KH2PO4</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>MgCl2</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&#160;</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&#160;</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 ...

The overall goal of this procedure is to isolate skeletal muscle mitochondria by differential centrifugation and determine the respiratory rates of mitochondrial respiratory chain complexes I, II and IV.This method can help answer key questions in the mitochondrial bio-energetic field such as diseases and syndromes associated with mitochondrial dysfunction, such as diverse neurological diseases, sepsis and age-related disorders. The main advantage of this technique is the absence most of the cytologic factors in the isolated mitochondrial preparation, which may interfere with the analysis of the mitochondria functions. Demonstrating the procedure will be Sandra Nansoz, the technician from my laboratory.To begin this procedure, obtain a beaker of skeletal muscle samples as outlined in the text protocol. Place the beaker on an ice bucket. Using fine scissors, mince the muscle into pieces one to two millimeters in size.Next, rinse the minced tissue twice with ice cold isolation buffer using 20 milliliters for each wash. Suspend the washed tissue in 10 milliliters of isolation buffer per gram of tissue and then add five milligrams protease-G tissue. Then place the beaker into an ice bath with a magnetic stirrer and stir for 10 minutes.After this, dilute the suspension with an additional 10 milliliters of isolation buffer supplemented with 0.2%de-fatted BSA per gram of tissue. Next, decant all of the isolation buffer from the beaker. Wash the tissue twice with ice cold isolation buffer supplemented with 0.2%BSA using 20 milliliters for each wash.Then, re-suspend the tissue in 10 milliliters of BSA supplemented isolation buffer per gram of tissue. Use a semi-automatic glass homogenizer with a loose-fitted pestle to homogenize the tissue. Next, transfer the homogenate to a centrifugation tube and centrifuge at 10, 000 times G for 10 minutes at four degrees Celsius.Using a serological pipette, discard the supernatant. Re-suspend the pellet with 10 milliliters of ice cold BSA supplemented isolation medium per gram of tissue. Centrifuge the suspension at 350 times G for 10 minutes at four degrees Celsius.After this, use a serological pipette to reserve the supernatant, discarding the pellet. Filter the supernatant into a beaker kept on ice through two layers of gauze. Transfer the filtered suspension to a centrifuge tube and centrifuge 7, 000 times G for 10 minutes at four degrees Celsius.Then discard the supernatant. Re-suspend the crude mitochondrial pellet in five milliliters of BSA supplemented isolation buffer per gram of tissue. Centrifuge this suspension 7, 000 times G for 10 minutes at four degrees Celsius.Next, discard the supernatant and re-suspend the pellet in 20 milliliters of wash buffer. Centrifuge at 7, 000 times G for 10 minutes at four degrees Celsius. Repeat this re-suspension and centrifugation process once more.After this, discard the supernatant and re-suspend the pellet in one milliliter of wash buffer. Determine the protein concentration using any standard method. Then, keep the concentrated mitochondrial suspension on ice until ready to perform respirometry assays.To begin, re-suspend the mitochondrial concentrate in the respiration buffer to a final concentration of 0.4 milligrams per milliliter mitochondrial protein. Next, aspirate the respiration medium from the air calibrated oxygraph chamber. Add 2.1 milliliters of isolated mitochondrial suspension.Insert a stopper to close the oxygraph chamber. Then, stir the mitochondrial suspension continuously at 700 RPM at 37 degrees Celsius. Record the cellular respiration at baseline for three to five minutes until a stable oxygen flux signal is achieved.To begin complex I-dependent respiration, inject 12.5 microliters of 0.8 molar malate into an oxygraph chamber prepared with the isolated mitochondrial suspension through the stopper's titanium injection port. Then, inject 10 microliters of two molar glutamate. Record cellular respiration for three to five minutes until a stable oxygen flux signal is achieved.After this, inject 10 microliters of 0.05 molar ADP into the chamber through the injection port. Record cellular respiration until the oxygen flux signal increases then decreases and stabilizes. For the complex II-dependent respiration, inject two microliters of 0.2 millimolar rotenone into an oxygraph chamber prepared with isolated mitochondrial suspension.Record the cellular respiration until a stable oxygen flux signal is achieved. Then, inject 20 microliters of one molar succinate. Record cellular respiration until a stable oxygen flux signal is achieved.After this, inject 10 microliters of 0.05 molar ADP into the chamber through the injection port. Record cellular respiration until the oxygen flux signal increases then decreases and stabilizes. For the complex IV-dependent respiration, inject 2.5 microliters of 0.8 millimolar ascorbate into an oxygraph chamber prepared with isolated mitochondrial suspension.Next, immediately inject 2.5 microliters of 0.2 molar TMPD and 10 microliters of 0.05 molar ADP. Record cellular respiration until a stable oxygen flux signal is achieved. Then, inject 10 microliters of one molar sodium azide.Record cellular respiration until the oxygen flux signal decreases, then stabilizes. In this study, intact mitochondria are isolated from skeletal muscle. Complex I-dependent respiratory rates are then measured via high resolution respirometry.As can be seen, the mitochondrial respiration rate increases immediately upon the addition of ADP. The respiration rate decreases after ADP depletion to levels similar to those observed before the addition of ADP. This indicates that the ratio of state 3 and state 4 known as the respiratory control ratio, or RCR, is high and that mitochondrial integrity is well preserved during the isolation procedure.Complex II-dependent respiratory rates are then observed by inhibiting complex I-respiration with rotenone and injecting the complex II-substrate succinate. ADP is then added, resulting in an immediate increase in respiration. Once ADP is depleted, respiration decreases to levels similar to those observed before the addition of ADP, indicating a high RCR, in that the mitochondrial integrity is well preserved during isolation.Complex IV-dependent respiration rates are observed by adding ascorbate, TMPD and ADP. Sodium azide is then added to inhibit complex IV.The difference in oxygen consumption before and after the addition of sodium azide is interpreted as the real complex IV respiration. The high complex IV-dependent respiration rate observed in state 3 indicates that mitochondrial integrity is wel preserved during the isolation procedure.Once mastered, this technique can be done in less than one and a half hours if it is performed properly. While attempting this procedure, it is important to remember to perform a calibration of the oxygen sensors in advance prior to homogenization. Following this procedure, other methods like measurements of cellular ADP content, mitochondrial membrane potential, ATP synthase enzymatic activity as substrate levels can be performed in order to answer additional questions like whether changes in respiratory rates are due to lack of mitochondrial substrate, membrane potential, or reduced ATP synthase enzymatic activity.After watching this video, you should have a good understanding of how to isolate mitochondria from a skeletal muscle by differential centrifugation and how to measure respiratory rates of mitochondrial complexes I, II and IV using high resolution respirometry. After its development, this technique paved the way for researchers in the field of mitochondrial energy metabolism to explore mitochondria energy function in fields such as genetic mitochondrial diseases, myopathies, ischemia reperfusion injury in isolated mitochondria. Don't forget that working with antimycin A, rotenone, sodium azide and FCCP can be extremely hazardous.And precautions such as wearing gloves and laboratory coats should always be taken while performing this procedure.

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17.8K vistas.  Inselspital, Bern University Hospital. The overall goal of this procedure is to isolate skeletal muscle mitochondria by differential centrifugation and determine the respiratory rates of mitochondrial respiratory chain complexes I, II and IV.This method can help answer key questions in the mitochondrial bio-energetic field such as diseases and syndromes associated with mitochondrial dysfunction, such as diverse neurological diseases, sepsis and age-related disorders. The main advantage of this technique is the absence most of the ...