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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</ol>

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。 骨骼肌线粒体隔离往往需要苛刻的方法从周围的结缔组织和结构蛋白7,16解放线粒体。因此，线粒体膜可以成为打乱，从而损害线粒体的 O 2消耗的活性（以及因此的精度）在随后respiromeTRIC检测。由包括组织使用刀片组织切除后到先前描述的方案7切碎的额外步骤，一个显著降低转子速度为电动机驱动的均化是可能的（80转与1600转）。这些温和的均质化和再悬浮技术导致线粒体胎膜完整（92.5±2.0％）的高百分比，如从下面的分离过程中的柠檬酸合酶活性进行评估。我们的发现与Asmann 等人 17，以下从人骨骼肌隔离90-95％的完整线粒体制剂之间谁报告一致。要注意的是测量柠檬酸合酶活性是更适合于测量线粒体膜的物理完整性而不应被用来评估分离线粒体的功能完整性是重要的。另一方面，O 2消耗测定法和测定RCR从这些测定应当用于MEA确保分离线粒体1的功能 。此外，RCR也可以用作耦合线粒体的一个指标。因此，一个典型的范围从丙酮酸/苹果酸盐法8，并从各种呼吸计测定法获得的高RCR值内OCR和线粒体状态表明从该协议得到的线粒体是紧耦合的，高功能。 完好和纯线粒体隔离具有respirometic检测具有重要意义。非线粒体蛋白质在最终线粒体制剂的污染可导致加载不等量的线粒体蛋白质，每孔中的 O 2消耗的测量，从而减小中间实验任何比较的精度来执行使用线粒体不同的制剂。此外，线粒体制剂与其它呼吸的细胞器的污染（例如，过氧化物酶）CAñ进一步降低氧气消耗量测量的准确度。分离过程期间的内线粒体蛋白，COXIV，以及缺乏胞质（和潜在的核）蛋白，GAPDH，在我们的孤立线粒体制备以下免疫印迹的显着条带的存在指示来自非线粒体来源少污染的。 要注意的是，尽管纯化线粒体制剂的隔离是相对于上述重要，线粒体完整性的维持是至关重要的呼吸测定法是重要的。线粒体外膜可以分离过程中很容易损坏，从而导致细胞色素c的逃逸到隔离缓冲器，从而成为速率期间的 O 2消耗和ATP合成18限制性的。因此，分离线粒体的完整性，可以进一步通过定量细胞色素评估在线粒体制备（步骤2.15，前再悬浮）并在分离的线粒体与Western印迹的上清欧共体蛋白的表达。细胞色素c的不应存在于线粒体制备上清液如果线粒体膜是完整的以下的分离方法。 所有步骤这个协议是重要的，但有三个关键步骤，这一协议。首先，红骨骼肌用三种不同刀片（步骤2.4）的切碎是关键的，因为该步骤允许在均质化步骤（步骤2.11）更慢的转子速度。其次，对冰或冷冻缓冲区很多步骤的性能是至关重要的，以保持在静止状态下，线粒体的基于微孔板检测呼吸速率之前。最后，将线粒体沉淀成IBM2的温和再悬浮是极其重要的。温和混合维持线粒体完整性，这对于准确RESP关键irometic检测。 这种技术的一些限制值得一提。首先，需要的设备（例如，超离心，马达驱动的均化器等 ），以执行分离过程可能是昂贵的。另外，需要多个质量控制方法，以确保清洁和完整的线粒体。然而，一旦研究人员精通分离过程中，高品质的线粒体产生了可在多种测量，包括被使用，但不限于:Western印迹，酶测定法，RT-PCR，PCR，H 2 O 2分析和高通量微量呼吸测量。要注意，此分离步骤得到了优化和修改后用于小批量的小鼠骨骼肌和谨慎试图从一个给定的组织来自相同物种的应用分离技术时，应采取这一点很重要（甚至来自不同物种的相同组织）到其他组织/特定上课。为获得最佳结果，检索文献努力寻找最好的隔离方法的预期的组织/种类时将是最好的选择。

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><tbody><tr><td>Essentially Fatty</td><td>Sigma Aldrich</td><td>A6003</td><td>N/A</td></tr><tr><td>Acid Free- BSA</td><td></td><td></td><td></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&lt;br/&gt; White Crystals or Crystalline Powder&lt;br/&gt; &amp;ge;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&amp;nbsp;</td><td>N/A</td></tr><tr><td>Sodium deoxycholate</td><td>Sigma Aldrich</td><td>D6750&amp;nbsp;</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>Halt Protease &amp;amp; Phosphatse Inhibitor Cocktail</td><td>Thermo Scientific</td><td>1861284</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>1.5 ml microcentrifuge tubes with screw cap</td><td>Thermo Scientific</td><td>3474</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Zirconium Oxide beads</td><td>Fisher Scientific</td><td>C9012112</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></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>Peroxidase conjugated affinipure Donkey, Anti Rabbit IgG (H+L)</td><td>Jackson ImmunoResearh</td><td>711-035-152</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Peroxidase conjugated affinipure Goat, Anti Mouse IgG (H+L)</td><td>Jackson ImmunoResearh</td><td>115-001-003</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></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&amp;nbsp;</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 &amp;deg;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, &amp;ge;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>L-Glutamic acid</td><td>Sigma Aldrich</td><td>G1251</td><td>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><td></td><td></td><td></td><td></td></tr><tr><td>Palmitoyl L-carnitine chloride</td><td>Sigma Aldrich</td><td>P1645</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Oligomycin A, &amp;ge; 95% (HPLC)</td><td>Sigma Aldrich</td><td>75351</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Carbonyl cyanide 4-(trifluoromethoxy)</td><td>Sigma Aldrich</td><td>C2920</td><td>Store at 2-8 &amp;deg;C</td></tr><tr><td>phenylhydrazone</td><td></td><td></td><td></td></tr><tr><td>&amp;ge;98% (TLC), powder [FCCP]</td><td></td><td></td><td></td></tr><tr><td>Antimycin A from streptomyces sp.</td><td>Sigma Aldrich</td><td>A8674</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Adenosine 5&amp;prime;-diphosphate monopotassium salt dehydrate [ADP]</td><td>Sigma Aldrich</td><td>A5285</td><td>Store at -20 &amp;deg;C, to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td></td><td></td><td></td><td></td></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 GAPDH和COXIV表达在分离线粒体和整个骨骼肌组织溶解液。 GAPDH是位于细胞溶质中的蛋白质，但也可以在转位后的细胞核中，而COXIV是位于线粒体内膜的蛋白质。 GAPDH和COXIV的表达是在整个TISS明显UE裂解液。在分离线粒体，COXIV的表达观察到，而GAPDH只有微弱的频带是显而易见的。这些结果表明良好线粒体隔离，由非线粒体成分的分离过程期间小的污染。 图3是从线粒体使用此协议中分离并用微孔板基于O 2的消耗技术执行进行O 2的消耗速率（OCR）对时间的一个耦合测定（10mM的丙酮酸/ 5mM的苹果酸盐）的代表性跟踪。每个小组代表O 2的消费所描述的机遇与威廉姆斯13种不同的线粒体状态。第一面板代表基底的 O 2消耗，或国家2.第二面板，注射的ADP后，表示最大连接呼吸，或国家3.第三面板，注射的寡霉素A（复合物V的抑制剂），represe后NTS呼吸由于质子漏，或国家40。第四盘，注射FCCP后，表示极大的解偶联呼吸作用，或国家3U。最后，第五面板，注射抗霉素A的后，表示氧化呼吸的抑制。呼吸控制率（RCR），线粒体功能1的量度，被除以状态3由国家的4o。 表4报告的平均RCR值，可以从这个协议的线粒体收率进行替代衬底耦合测定。重要的是，O 2的消耗跟踪和高RCR值表示高度运作，加上线粒体。本文所报道的RCR值是高于或类似比先前报道使用在小鼠骨骼肌7,14,15类似分离方案，从而加剧在此过程中分离出的线粒体的高品质。 图1:柠檬酸合成酶的活性 。柠檬酸合酶的酶活性作为非超声处理超声处理和线粒体用制剂分光光度法测定。值表示为平均值±SEM表示。 ** P &lt;0.01。数据表示n = 3的配对生物学重复，并表示相对于毫克的蛋白质。 <img alt="图1" src="/files/ftp_upload/53217/53217fig1.jpg" /> 图2:细胞质和分离线粒体和整个组织裂解线粒体蛋白表达两者分离线粒体和整个组织裂解物进行免疫印迹以COXIV和GAPDH的抗体。线粒体蛋白质，COXIV，是明显的两个样品中，但是GAPDH只是依稀显而易见的，在分离线粒体。中日缺乏突出的GAPDH带Ë分离线粒体表明一点污染从隔离过程中的非线粒体来源。 <img alt="图3" src="/files/ftp_upload/53217/53217fig3.jpg" /> 图3:耦合测定与线粒体从小鼠骨骼肌中分离代表跟踪的10mM丙酮酸/ 5mM的苹果酸盐耦合线粒体呼吸描记法如通过氧消 ​​耗多井测量来确定。值表示为平均值±标准差。每孔装线粒体蛋白是2.5微克。 OCR = O 2消耗率; ADP =二磷酸腺苷;寡=寡A; FCCP =羰基氰- 4 - （三氟甲氧基）苯腙;抗A =抗霉素A. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td rowspan="2"> 试剂 </td><td> 股票集中度 </td><td><strong&gt; MW </td><td> 最终体积 </td><td> 大规模增加 </td></tr><tr><td> （M）的</td><td> （克/摩尔） </td><td> （毫升） </td><td> （G） </td></tr><tr><td>三/盐酸，pH值7.4 </td><td> 2 </td><td> 157.56 </td><td> 250 </td><td> 78.8 </td></tr><tr><td>三/盐酸，pH值7.4 </td><td> 1 </td><td> 157.56 </td><td> 250 </td><td> 39.39 </td></tr><tr><td> KCL </td><td> 1 </td><td> 74.55 </td><td> 500 </td><td> 37.28 </td></tr><tr><td>三基地</td><td> 1 </td><td> 121.14 </td><td> 100 </td><td> 12.11 </td></tr><tr><td> EDTA，pH为8 </td><td> 0.5 </td><td> 416.2 </td><td> 100个毫升（在1M Tris碱） </td><td> 20.81 </td></tr><tr><td> EGTA，pH 7.2的</td><td> 0.1 </td><td> 380.35 </td><td> 100（在1M Tris碱） </td><td> 3.801 </td></tr></tbody></table> 表1:股票方案准备。 <table b为了="0"的cellpadding ="0"CELLSPACING ="0"&gt; <tbody><tr><td rowspan="2"> 试剂 </td><td> 股票集中度 </td><td> 大规模增加 </td><td rowspan="2"> 最后摩尔浓度/百分比 </td></tr><tr><td> （M）的</td><td> （g或毫升） </td></tr><tr><td>蔗糖</td><td> - </td><td> 11.47克</td><td> 67毫米</td></tr><tr><td>三/盐酸，pH值7.4 </td><td> 2 </td><td> 12.5毫升</td><td> 50毫米</td></tr><tr><td> KCL </td><td> 1 </td><td> 25毫升</td><td> 50毫米</td></tr><tr><td> EDTA / Tris碱，pH值8 </td><td> 0.5 </td><td> 10毫升</td><td> 10毫</td></tr><tr><td>从本质上讲FA免费BSA </td><td> - </td><td> 1克</td><td> 0.20％ </td></tr><tr><td colspan="4"> pH 7.4的500毫升:分装50个ml和冷冻</td></tr></tbody></table> 表2:隔离缓冲线粒体1（IBM1）的制备。<table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td rowspan="2"> 试剂 </td><td> 股票集中度 </td><td> 大规模增加 </td><td rowspan="2"> 最后摩尔浓度/百分比 </td></tr><tr><td> （M）的</td><td> （g或毫升） </td></tr><tr><td> D-甘露醇</td><td> - </td><td> 10.9克</td><td> 200毫米</td></tr><tr><td>蔗糖</td><td> - </td><td> 7.188克</td><td> 70毫米</td></tr><tr><td> EGTA / Tris碱</td><td> 0.1 </td><td> 15毫升</td><td> 5毫米</td></tr><tr><td>的Tris-HCl，pH 7.4中</td><td> 1 </td><td> 3毫升</td><td> 10毫</td></tr><tr><td colspan="4"> pH为7.4，将300ml:分装15个ml和冷冻</td></tr></tbody></table> 表3:隔离缓冲线粒体2（IBM2）的制备。 <table border="0" cellpadding="0" cellspacing="0"><tbody&gt; <tr><td> 基材中 </td><td> 终浓度 </td><td> RCR </td></tr><tr><td>丙酮酸/马拉特</td><td> 10毫米/ 10毫米</td><td> 16.2±4.6 </td></tr><tr><td>琥珀酸/鱼藤酮</td><td>的10mM / 2μM </td><td> 10.6±3.8 </td></tr><tr><td>棕榈左旋肉碱/马拉特</td><td> 40微米/ 1毫米马拉特</td><td> 6.7±0.6 </td></tr><tr><td>谷氨酸/马拉特</td><td>的10mM / 10mM的</td><td> 8.6±0.4 </td></tr></tbody></table> 表4:对通过第一测量氧气消耗率与微孔板基于透气性检测，随后通过将最大加上呼吸决定再加线粒体呼吸测定呼吸控制比率呼吸控制比率 （ADP刺激的国3）呼吸，由于质子漏（寡的响应国家40）。值表示为箱一个±SE。每孔装线粒体蛋白2.5微克丙酮酸/苹果酸和琥珀酸/鱼藤酮测定，和3.5微克每口井的线粒体蛋白的棕榈酰左旋肉碱/苹果酸和谷氨酸/苹果酸测定的。

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

Nanyang Technological University

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

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

科研

发育生物学

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, mitochondria exist in a series of complex morphologies, ranging from small oval organelles to a broad, reticulum-like network. Understanding how the mitochondrial reticulum expands and develops in response to diverse stimuli such as alterations in energy demand has long been a topic of research. A key aspect of this growth, or biogenesis, is the import of precursor proteins, originally encoded by the nuclear genome, synthesized in the cytosol, and translocated into various mitochondrial sub-compartments. Mitochondria have developed a sophisticated mechanism for this import process, involving many selective inner and outer membrane channels, known as the protein import machinery (PIM). Import into the mitochondrion is dependent on viable membrane potential and the availability of organelle-derived ATP through oxidative phosphorylation. Therefore its measurement can serve as a measure of organelle health. The PIM also exhibits a high level of adaptive plasticity in skeletal muscle that is tightly coupled to the energy status of the cell. For example, exercise training has been shown to increase import capacity, while muscle disuse reduces it, coincident with changes in markers of mitochondrial content. Although protein import is a critical step in the biogenesis and expansion of mitochondria, the process is not widely studied in skeletal muscle. Thus, this paper outlines how to use isolated and fully functional mitochondria from skeletal muscle to measure protein import capacity in order to promote a greater understanding of the methods involved and an appreciation of the importance of the pathway for organelle turnover in exercise, health, and disease.

Mitochondria are key metabolic organelles that exhibit a high level of phenotypic plasticity in skeletal muscle. The import of proteins from the cytosol is a critical pathway for organelle biogenesis, essential for the expansion of the reticulum and the maintenance of mitochondrial function. Therefore, protein import serves as a barometer of cellular health.

骨骼肌线粒体蛋白质导入能力的测定

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, ...

生物化学

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

线粒体的制备及透气性评估，从骨骼肌组织通过穿刺活检获得的分离

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

医学

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 ...

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 ...

<meta charset="utf8"></head><body>从啮齿动物中分离线粒体以进行高质量的生物能量研究

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry to study metabolic impairments and disease. Consistent mitochondrial quality is necessary to properly investigate mitochondrial physiology and bioenergetics; however, many different published isolation methods are available for researchers. Although different experimental strategies require different isolation methods, the basic principles and procedures are similar. This protocol details a method capable of extracting well-coupled mitochondria from a variety of tissue sources, including small animals and cells. The steps outlined include organ dissection, mitochondrial purification, protein quantification, and various quality control checks. The primary quality control metric used to identify high-quality mitochondria is the respiratory control ratio (RCR). The RCR is the ratio of the respiratory rate during oxidative phosphorylation to the rate in the absence of ADP. Alternative metrics are discussed. While high RCR values relative to their tissue source are obtained using this protocol, several steps can be optimized to suit the individual needs of researchers. This procedure is robust and has consistently resulted in isolated mitochondria with above-average RCR values across animal models and tissue sources.

<meta charset="utf8"></head><body>作者聚焦:揭示线粒体磷酸钙在心力衰竭和生物能量学中的作用

Bioenergetic and metabolomic studies on mitochondria have revealed their multifaceted role in many diseases, but the isolation methods for these organelles vary. The method detailed here is capable of purifying high-quality mitochondria from multiple tissue sources. Quality is determined by respiratory control ratios and other metrics assessed with high-resolution respirometry.

从啮齿动物心脏组织中稳健地分离线粒体 

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

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

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使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

骨骼肌线粒体蛋白质导入能力的测定

线粒体的制备及透气性评估，从骨骼肌组织通过穿刺活检获得的分离

成人和胚胎骨骼的肌肉Microexplant文化和骨骼肌肉干细胞的分离

骨骼肌的线粒体分离

纯化的骨骼肌的先觉者

从啮齿动物心脏组织中稳健地分离线粒体

从啮齿动物心脏组织中稳健地分离线粒体

从啮齿动物心脏组织中稳健地分离线粒体

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。