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 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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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

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 ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

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 ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

The goal of this protocol is to measure the effect of glucose-mediated changes to mitochondrial respiration in the presence of natural compounds on intact 832/13 beta cells using high-resolution respirometry.

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role ...