The Fralin Life Science Research Institute and The Metabolic Phenotyping Core at Virginia Tech supported this work.

经批准的协议下进行的机构动物护理动物的研究和利用委员会在弗吉尼亚理工学院和州立大学。 1.设置（时间:〜45分钟） <ol><li> 0.25％胰蛋白酶，分离缓冲液为线粒体（IBM）的1和IBM2在37℃水浴中解冻冷冻的商店。 </li><li>冲洗玻璃器皿和在70％乙醇接着通过高纯度水解剖仪器。 </li><li>由4部分IBM1稀释1份胰蛋白酶准备从0.25％胰蛋白酶股票0.05％胰蛋白酶溶液。 </li><li>混合蛋白酶和磷酸酶抑制剂混合物用细胞裂解缓冲液:在1.5ml微量离心管中与螺旋盖（1 100比）。 </li><li>等分试样5 ml的0.05％胰蛋白酶（5毫升/小鼠）入15ml塑料管。 </li><li>设置电机驱动组织匀浆80 RPM。 </li></ol> 2.隔离骨骼肌线粒体（时间:〜90分钟） <ol><li>安乐死鼠标的CO 2 </suB&gt;吸入，颈椎脱位。 </li><li>除去从股四头肌和腓肠肌，其中包括如在下面的步骤中描述的比目鱼（〜75-100 mg总）红肌: <ol><li>剥离朝向鼠标皮肤。 </li><li>去除脂肪垫在股四头肌原点。切附加到用细尖的剪刀髌骨股四头肌腱。 注意:股四头肌由其上股骨近端的前部解剖位置识别。 </li><li>慢慢地剪断骨和四头肌之间的腱膜，同时也避免了股动脉，解放从骨四头肌。切细尖的剪刀肌腱在原点上股骨解放股四头肌，并将在冷冻PBS股四头肌。 </li><li>除去可见的脂肪组织过用剪刀股四头肌。翻转股四头肌过这样被覆盖在股骨肌肉的部分正面临ü页。打开股四头肌在一个扇形运动镊子。从每个叶用细尖的剪刀和地点，含5毫升冷冻IBM1的烧杯中取出两个可见的红肌部。 注意:股四头肌肌肉表现为两个波瓣的红肌位于每个波瓣的横向边缘的条带。 </li><li>切开皮肤覆盖跟腱用细尖的剪刀和剥离对鼠标的皮肤。切的露出跟腱和剥离肌肉朝向鼠标的主体。 </li><li>切牛筋用细尖的剪刀解放腓肠肌和地点连接到其肌腱冷冻PBS腓肠肌股骨外侧和内侧髁。 </li><li>翻转腓肠肌和剥离比目鱼肌并放入含有5毫升冷冻IBM1的烧杯中。风扇打开腓肠肌。卸下三个视觉红肌部（两个侧面和一个内侧表面的红色条）用细尖剪S和它们转移到含有5毫升冷冻IBM1的烧杯中。 </li></ol></li><li>从小鼠（如步骤2.2中所述）的另一条腿删除其它〜75-100毫克肌红并放入1.5ml微量离心管中以蛋白酶和磷酸酶抑制剂混合物和细胞裂解缓冲液（50mM Tris-盐酸，1毫摩尔EDTA，150毫摩尔NaCl，1％十二烷基硫酸钠，0.5％脱氧胆酸钠，1％聚氧乙烯（9）壬基苯基醚，支链。立即闪冻在液氮中在蛋白质印迹以后使用该第二样品（参见步骤6.1）。 </li><li>放置在冰上预冷，平板塑料表面在一个大的桶。倒一滴IBM1的在塑料表面使用镊子将所有的切片红肌（步骤2.2.4和2.2.7）的IBM1液滴。切碎的肌肉组织，使用3个单刃刀片通过改变剃须刀，每40秒2分钟。 </li><li>通过保持在塑料表面上次转移组织糜到一个新的烧杯用5ml新鲜IBM1Ë烧杯和刮肌成用刀片的烧杯中。取该溶液通过100微米的细胞滤网置于50ml锥形管漏它。 </li><li>吸干一个微妙的任务雨刷组织，然后将其转移到5毫升的0.05％胰蛋白酶溶液。使用镊子从任务雨刮器去除组织。 </li><li>冰上孵育的肌肉组织中的0.05％胰蛋白酶溶液30分钟。 </li><li>在200×g离心旋转15毫升锥形管含有胰蛋白酶/肌肉混合物3分钟，在4℃。 </li><li>倾胰蛋白酶上清到废物容器和悬浮颗粒用3毫升冰冷的IBM1。转移组织到一个45毫升玻璃均化管中。冲洗的15毫升锥形管与另一1.5 ml的IBM1收集任何残留的样品并且将其添加到玻璃匀浆管中。 </li><li>放置玻璃匀浆管放入烧杯或塑料容器填充中途用冰使玻璃匀浆最小移动烧杯内。 </li><li&gt;匀化组织/ IMB1混合物与以80rpm连接到一个马达驱动的组织匀浆用10经过聚四氟乙烯杵。保存每个通行证〜2秒的底部。 </li><li>该组织匀浆转移到一个新的15 mL锥形管冲洗玻璃匀浆管6.5毫升IBM1的。倒入IBM1到15毫升锥形管含组织匀浆。 </li><li>旋的15毫升锥形管中，在700克10分钟，在4℃。轻轻倒出上清液到玻璃高强度离心管并弃沉淀。 </li><li>旋转从上述步骤的上清液在8,000rpm离心10分钟，在4℃。 </li><li>除去IBM1上清，通过缓慢加入500μl的IBM2的重悬浮沉淀。切断一个枪头的末端轻轻均质沉淀在IBM2与混合和搅拌运动。添加另一个4.5毫升IBM2向混合物后沉淀完全暂停。 注意:避免过度研磨而破大颗粒件为T他的可能损坏线粒体膜。 </li><li>旋转的IBM2 /组织匀浆在8000离心10分钟，在4℃。 注意:此步骤可以重复在一个干净的高强度离心管分离线粒体的更准确的蛋白质定量在步骤4中，因为残留的BSA可能有助于总蛋白质含量。 </li><li>去除上清，轻轻地，而是完全通过增加IBM2两个25μl递增用枪头的点切断重悬浮颗粒。轻轻搅拌，每加入25微升IBM2后混合沉淀。把这个线粒体股票冰上。 </li></ol> 3.同质化的全组织裂解液（时间:〜45分钟;可完成在第7步） <ol><li>取出肌肉从被放置在液氮从步骤2.3和在室温下孵育直到完全解冻。 </li><li>剁碎的组织为〜30秒在冰上用细尖的剪刀。 </li><li>加入Zirco两个100微升勺鎓氧化物珠到1.5 ml离心管与步骤3.2包含组织糜螺丝帽。均化在使用两个到3 5分钟循环的4-6（介质设置）一个速度设定珠磨机组织匀浆组织。 </li><li>旋转从步骤3.3，混合物在12000×g离心10分钟，在4℃。删除使用自旋和转印之后1,000微升枪头上清液至1.5 ml微量离心管中。弃沉淀并将上清液在冰上。 </li></ol> 4.蛋白质测定法（时间:约30分钟） <ol><li>确定根据制造商的规格使用BCA蛋白测定试剂盒的整个组织溶解液（步骤3.4）和线粒体股票（步骤2.17）的蛋白质浓度。 </li></ol> 5.线粒体膜的完整性;柠檬酸合成酶（CS）活动（时间:约15分钟） <ol><li>让两个独立的1:20稀释线粒体股票的高纯度水。加的Triton 100-X以一个样品的0.1％和超声处理它设定在一个低水平（765瓦，2幅）。留在冰等稀释。返回样品冰超声处理后。 </li><li>与执行柠檬酸合酶测定法既非超声处理并使用分光光度测定法如先前所述11,12-超声处理线粒体稀释。 </li><li>通过使用下面的方程确定完整的线粒体膜的百分比: （非超声CS活动÷超声CS活动）* 100 </li></ol> 100- N（％，从上面获得） 6.线粒体提取质量; Western印迹（时间:根据实验室协议） <ol><li>如先前描述的12对整个组织溶解液和分离线粒体进行Western印迹。 注意:使用对GAPDH初级抗体（1:1000稀释在5％BSA / TBS-T）和COXIV（1:1000稀释在5％脱脂乳/ TBS-T），然后加入抗山羊和兔二抗，分别为（1:10,000）。 </li></ol> 7.透气性试验运行（时间:〜90分钟） <ol><li>执行使用基于微孔板O 2消耗量测量技术如前所述所需的透气性测定法（见同伴文章和罗杰斯等人8）。 </li><li>除以3国国家元首40（RCR）或除国家3U由国家40确定呼吸控制率（RCR）。从点至点痕迹确定RCR当使用两中间（平均值）点，或最高（状态3）和费用最低（状态40）。 </li></ol>

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George Rogers is an employee of Seahorse Bioscience that produces the instrument for which this protocol was modified.

本文所提出的方法提供了从最小量的小鼠骨骼肌（〜75-100毫克）线粒体分离方法的详细描述。这种隔离方法能够产生高功能，纯粹的线粒体（〜450微克）就证明了氧气的消耗速率，RCR值，最大的柠檬酸合成酶的活性和免疫蛋白的表达。重要的是，可用于多种respirometirc测定用微孔板基于O 2的消耗技术，它允许高通量从这个过程中分离线粒体。 骨骼肌线粒体隔离...

The primary function of mitochondria is to produce ATP from oxidative phosphorylation. However, mitochondria have many other important cellular functions including but not limited to: the production and detoxification of reactive oxygen species, the regulation of cytoplasmic and mitochondrial calcium, organelle trafficking, ionic homeostasis, and involvement in apoptosis1,2. Therefore, it is not surprising that dysfunctional mitochondria play a role in many disease pathologies, such as aging, neurodegenerative diseases, cardiovascular disease, cancer, obesity, and diabetes3,4. Importantly, skeletal muscle mitochondria specifically are involved in many of these pathologies3-5.
Mitochondrial respiration assays using isolated mitochondria allow for the assessment of electron transport chain and oxidative phosphorylation function, and the determination of mechanism(s) of action of drugs and proteins that modulate metabolism. Mitochondrial isolation procedures exist for multiple tissue and cell types for a variety of species6,7. However, these procedures often require large quantities of tissue/cells for a high quality mitochondria yield necessary for classic respirometric assays. 
Microplate based respirometirc assays allow for high throughput measurements using minimal quantities of isolated mitochondria, often just several &#181;g per well8. Therefore, we present a modification of previously published methods7 to allow for high quality mitochondria to be isolated from smaller quantities of mouse skeletal muscle for use in microplate based respirometirc assays. In addition, methods are provided to establish the quality of the mitochondrial isolation preparation and the integrity of the mitochondrial membranes. Given that skeletal muscle mitochondria are involved in many pathological conditions, the measurement of O2 consumption in mechanistically driven studies is becoming more prevalent in biomedical research9,10.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Essentially Fatty</td>
			<td rowspan="2">Sigma Aldrich</td>
			<td rowspan="2">A6003</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
			<td>Acid Free- BSA</td>
		</tr>
		<tr>
			<td>Tris/HCl</td>
			<td>Promega</td>
			<td>H5123</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>KCL</td>
			<td>Sigma Aldrich</td>
			<td>P9541</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Promega</td>
			<td>H5135</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>E6511</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma Aldrich</td>
			<td>E4378</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S7903</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>D-Mannitol</td>
			<td>Sigma Aldrich</td>
			<td>63559</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Scientific</td>
			<td>25200-056</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Sodium Chloride 
			White Crystals or Crystalline Powder 
			&#8805;99.0 %</td>
			<td>Fisher Scientific</td>
			<td>BP3581</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Sigma Aldrich</td>
			<td>L3771&#160;</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma Aldrich</td>
			<td>D6750&#160;</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Polyoxyethylene (12) nonylphenyl ether, branched</td>
			<td>Sigma Aldrich</td>
			<td>238651</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Single Edge Razor Blades</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Falcon- 100 uM Nylon Cell Strainers</td>
			<td>Fisher Scientific</td>
			<td>352360</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td rowspan="2">Halt Protease &#38; Phosphatse Inhibitor Cocktail</td>
			<td rowspan="2">Thermo Scientific</td>
			<td rowspan="2">1861284</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">1.5mL microcentrifuge tubes with screw cap</td>
			<td rowspan="2">Thermo Scientific</td>
			<td rowspan="2">3474</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Zirconium Oxide beads</td>
			<td rowspan="2">Fisher Scientific</td>
			<td rowspan="2">C9012112</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>GAPDH antibody (1D4)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-59540</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Anti- COXIV antibody</td>
			<td>Cell Signaling</td>
			<td>4844s</td>
			<td>Any mitochondrial inner membrane protein will suffice</td>
		</tr>
		<tr>
			<td rowspan="2">Peroxidase conjugated affinipure Donkey, Anti Rabbit IgG (H+L)</td>
			<td rowspan="2">Jackson ImmunoResearh</td>
			<td rowspan="2">711-035-152</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Peroxidase conjugated affinipure Goat, Anti Mouse IgG (H+L)</td>
			<td rowspan="2">Jackson ImmunoResearh</td>
			<td rowspan="2">115-001-003</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Triton-X100</td>
			<td>Sigma Aldrich</td>
			<td>X100</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit&#160;</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Pyruvic Acid, 98%</td>
			<td>Sigma Aldrich</td>
			<td>107360</td>
			<td>Store at 4&#176;C,pH to 7.4 with KOH prior to use in respirometric assay</td>
		</tr>
		<tr>
			<td>Succinic Acid</td>
			<td>Sigma Aldrich</td>
			<td>S9512</td>
			<td>Store at room temperature, pH to 7.4 with KOH prior to use in respirometric assay</td>
		</tr>
		<tr>
			<td>L(-) Malic Acid, BioXtra, &#8805;95%</td>
			<td>Sigma Aldrich</td>
			<td>M6413</td>
			<td>Store at room temperature, to 7.4 with KOH prior to use in respirometric assay</td>
		</tr>
		<tr>
			<td rowspan="2">L-Glutamic acid</td>
			<td rowspan="2">Sigma Aldrich</td>
			<td rowspan="2">G1251</td>
			<td rowspan="2">Store at room temperature, to 7.4 with KOH prior to use in respirometric assay, to 7.4 with KOH prior to use in respirometric assay</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Palmitoyl L-carnitine chloride</td>
			<td>Sigma Aldrich</td>
			<td>P1645</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Oligomycin A, &#8805; 95% (HPLC)</td>
			<td>Sigma Aldrich</td>
			<td>75351</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Carbonyl cyanide 4-(trifluoromethoxy)</td>
			<td rowspan="3">Sigma Aldrich</td>
			<td rowspan="3">C2920</td>
			<td rowspan="3">Store at 2-8&#176;C</td>
		</tr>
		<tr>
			<td>phenylhydrazone</td>
		</tr>
		<tr>
			<td>&#8805;98% (TLC), powder [FCCP]</td>
		</tr>
		<tr>
			<td>Antimycin A from streptomyces sp.</td>
			<td>Sigma Aldrich</td>
			<td>A8674</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td rowspan="2">Adenosine 5&#8242;-diphosphate monopotassium salt dehydrate [ADP]</td>
			<td rowspan="2">Sigma Aldrich</td>
			<td rowspan="2">A5285</td>
			<td rowspan="2">Store at -20&#176;C, to 7.4 with KOH prior to use in respirometric assay</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma Aldrich</td>
			<td>R8875</td>
			<td>Store at room temperature</td>
		</tr>
	</tbody>
</table>

isolation of mitochondria from minimal quantities of mouse skeletal muscle for high throughput microplate respiratory measurements

功能失调骨骼肌线粒体起到与老化，肥胖和II型糖尿病观察改变的代谢作用。从分离的线粒体制剂线粒体呼吸计测定允许对线粒体功能的药物和能调节代谢蛋白质的作用机制（多个）的评估，以及判定。目前的隔离程序往往需要大量的组织得到必要的透气性测定高品质的线粒体。本文所提出的方法描述了如何高品质纯化线粒体（〜450微克），可以从最小量的小鼠骨骼肌（〜75-100毫克）分离为高通量呼吸测量使用。我们确定了我们的隔离方法产生92.5±2.0％的完整线粒体通过测量柠檬酸合成酶的活性分光光度法。此外，在分离线粒体Western blot分析结果在微弱的表达cytoso的LIC蛋白，GAPDH，和线粒体蛋白质，COXIV的健壮表达。由于没有在分离线粒体的一个突出的GAPDH频带的指示小污染从隔离过程期间非线粒体来源。最重要的是，O 2的消耗率与微板为基础的技术，并确定用于耦合呼吸计测定显示高度耦合的呼吸控制率（RCR）的测量（RCR;&gt; 6对于所有测定）和功能性线粒体。总之，增加了一个单独的切碎工序和显著减少先前报道的方法的电动机驱动的均化速度已允许的高品质和纯化的线粒体从较少量的小鼠骨骼肌，其导致高度耦合的线粒体，随着高功能呼吸作用的隔离在基于微孔板respirometirc检测。

柠檬酸合酶活性充当细胞膜的完整性，因为柠檬酸合酶位于线粒体内膜，并且因此不应该存在于线粒体中具有完整的膜悬浮液的测量。 图1表示非超声处理线粒体样品中的柠檬酸合酶活性带有超声处理相比样品相同的隔离。声波处理线粒体结果在统计学上显著增加柠檬酸合酶活性（P &lt;0.01）。重要的是，以下的隔离线粒体的92.5±2.0％是完整的。 图2描述了</...

Watch this Scientific Journal Video about Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements at JoVE.com

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

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.

从小鼠骨骼肌高通量微孔板呼吸测量最低数量的隔离线粒体

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

isolation-mitochondria-from-minimal-quantities-mouse-skeletal-muscle

Research

JoVE Journal

Biology

12.7K 次观看.  Virginia Tech. 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.

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

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.

成人和胚胎骨骼的肌肉Microexplant文化和骨骼肌肉干细胞的分离

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

科研

生物学

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.

骨骼肌的线粒体分离

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.

纯化的骨骼肌的先觉者

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.

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

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

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.

从人骨骼肌隔离血管源性多能前体

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

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

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.

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.

骨骼肌完整的线粒体的分离差速离心高分辨率测量呼吸运动

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

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

骨骼肌成纤维细胞外泌体的分离和表征

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

分离高质量鼠心房和心室肌细胞，同时测量 Ca2+ 瞬变和 L 型钙电流

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions can be converted back to NO in mammals through a two-step reduction mechanism: nitrate being reduced to nitrite (NO2-) mostly by oral commensal bacteria, then nitrite being reduced to NO by several mechanisms including via heme- or molybdenum-containing proteins. This reductive nitrate pathway contributes to enhancing NO-mediated signaling pathways, particularly in the cardiovascular system and during muscular exercise. The levels of nitrate in the body before such utilization are determined by two different sources: endogenous NO oxidation and dietary nitrate intake, principally from plants. To elucidate the complex NO cycle in physiological circumstances, we have examined further the dynamics of its metabolites, nitrate and nitrite ions, which are relatively stable compared to NO. In previous studies skeletal muscle was identified as a major storage organ for nitrate ions in mammals, as well as a direct source of NO during exercise. Therefore, establishing a reliable methodology to measure nitrate and nitrite levels in skeletal muscle is important and should be helpful in extending its application to other tissue samples. This paper explains in detail the preparation of skeletal muscle samples, using three different homogenization methods, for nitrate and nitrite measurements and discusses important issues related to homogenization processes, including the size of the samples. Nitrate and nitrite concentrations have also been compared across four different muscle groups.

We present protocols for three different methods for the homogenization of four different muscle groups of rat skeletal muscle tissue to measure and compare the levels of nitrate and nitrite. Furthermore, we compare different sample weights to investigate whether tissue sample size affects the results of homogenization.

用于硝酸盐和亚硝酸盐测量的大鼠骨骼肌匀浆的制备

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions ...