Name: isolation of intact mitochondria from skeletal muscle by differential centrifugation for high-resolution respirometry measurements
Uploaded: 2017-03-08T14:00:00
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 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 ...

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