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 ...

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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.