这项工作是由一个由美国国家眼科研究所（R01 EY12998）授予FH安德拉德支持。

1。缓冲区的制备<ol><li>开启离心机5804R，并设置为4 ° C。 Isotemp 3006D水浴，并设置转到37 ° C </li><li>肌肉隔离前的准备以下解决方案: <ul><li> PBS:磷酸盐缓冲液（PBS）药片溶解在蒸馏水（5片/升）。拌匀。 </li><li> PBS加10 mM的EDTA:要准备100毫升溶液，添加98毫升的PBS 2毫升500毫米EDTA。 </li><li> 8X线粒体缓冲液:蔗糖10.28克终浓度为0.6米，400毫克的自由脂肪酸的牛血清白蛋白（BSA）终浓度为0.8％，为160毫米的终浓度2.08克HEPES，pH值7.4和QS至50毫升，用蒸馏水。 </li><li>隔离缓冲1（IB1）:加入200μL500 mM的EDTA的终浓度为10毫米，0.392克终浓度为215毫米D -甘露醇，8X线粒体缓冲液，pH 1.25毫升至7.4和QS至10毫升，用蒸馏水。 </li><li>隔离缓冲2（IB2）:加入终浓度为3毫米，0.392克终浓度为215毫米D -甘露醇，8X线粒体缓冲液，pH 1.25毫升至7.4和QS 60至10μL500毫米EGTA毫升，用蒸馏水。 </li><li>实验缓冲液（EB）:加入终浓度为100μLMgCl 2的 500毫米的终浓度为5毫米，0.392克终浓度为215毫米D -甘露醇，25微升2.5毫米KH 2 PO 4 6.25微米，2个100毫米EGTAμL终浓度为20微米，1.25毫升线粒体缓冲液，pH为7.4和QS至10毫升，用蒸馏水。 </li></ul></li></ol> 2。肌肉隔离<ol><li>在冰桶中，把3个10毫升的烧杯中，波特Elvehjem组织磨床和其他一切必要的仪器/用品上冰。整个实验过程中的一切都必须保持冰冷。 </li><li>放置在冰桶和温水浴​​EB IB1和IB2完成时的场所。 </li><li>在三个10毫升的烧杯中，应增加以下解决方案: <ul><li>烧杯1:10毫升的PBS </li><li>烧杯2:10毫升的PBS / EDTA </li><li>烧杯3:3毫升IB1 </li></ul></li><li>杀死老鼠人道本地机构的动物护理和使用委员会批准。 </li><li>迅速隔离骨骼肌250-500毫克，在烧杯1冲洗，然后转移到烧杯2。 </li></ol> 3。同质化/线粒体隔离<ol><li>肌肉转移到烧杯3和精细直言不讳地用剪刀肌肉。 </li><li>传输的解决方案，以波特Elvehjem匀浆。均质使用机动杵（一台钻床，会做的工作）的10倍，在任何时候都保持在冰管的肌肉。 </li><li>转移匀浆在700 克 10分钟，在4 ° C预冷的2毫升离心管和离心机</li><li>上清转移到一个新的预冷的离心管。弃去沉淀。 </li><li>上清离心10分钟，在10500 克 ，4 ° C 。 </li><li>上清转移到一个新的预冷的离心管和标签SN1（上清1号）和肌肉类型。 </li><li>重悬在500μL的IB2颗粒。 </li><li>离心10分钟，在10500 克在4 ° C。 </li><li>上清转移到一个新的预冷的离心管和标签SN2（上清2）和肌肉类型。 </li><li>暂停在100μL的IB2最终线粒体颗粒。 </li><li>重新自旋最终线粒体悬浮在minifuge几秒钟。如果有颗粒，转移上清到一个新的预冷的离心管，弃去沉淀。 </li><li>确定蛋白浓度用Bradford检测8。 </li></ol> 4。电极校准注:在线粒体隔离期间完成电极校准。 <ol><li>加入100毫升蒸馏水至250毫升容量瓶中。大力搅拌20分钟，以平衡水与大气中的气体。 </li><li>添加几滴50％氯化钾Oxygraph电极。 </li><li>放置一个电极上的一小块蓝Rizla卷烟纸。 </li><li> PTFE膜放置一块卷烟纸的顶部。 </li><li>申请使用环撒施，然后放置在电极槽外圈内圈。 </li><li>连接电极和组装其他设备:较大的塑料环基地，线粒体室，水管。 </li><li> Oxygraph箱打开，并启动Oxygraph软件。 </li><li>平衡水线粒体室。 </li><li>搅拌棒和打开速度为60。 </li><li>开始一个新的实验。 </li><li>点击"校准"和液相校准框1。变温度至37 ° C，然后单击"确定"两次。 </li><li>当"覆盖"转变到"确定"点击它，打开氮气罐。 </li><li>广场一角相连氮气罐室，并建立零氧。点击"OK"时，从开关"覆盖。" </li><li>吸干水和线粒体商会的EB缓冲液加入500μL。 </li><li>密封腔与柱塞。 </li><li>如果使用多个盒子，重复步骤11-15校准盒2。 </li></ol> 5。线粒体呼吸<ol><li>开始一个新的实验，让它运行约1分钟，以稳定的信号。 </li><li>添加必要的线粒体悬液量（步长3.11），为150微克每一个分庭，标志事件，并让它运行1分钟。 </li><li>加入10μl的温暖250 mM/125 mM的谷氨酸/苹果酸终浓度为5 mM/2.5毫米。马克和让运行1分钟。这是定义为国家2。 </li><li>加入终浓度为150微米，马克7.5μL10 mM的ADP公司，并让运行了30秒。这被定义为国家3。 </li><li>添加另有10毫米ADP，马克7.5μL，让运行1.5分钟。 </li><li>加入终浓度为1微米，马克0.5μL冷10毫米寡，让运行3分钟。这被定义为国家4。 </li><li>加入终浓度为0.2微米，马克1μL0.1 mM的FCCP冷，让运行3分钟。这是定义为5国。 </li><li>完成后，结束实验，并保存。 </li><li>获取使用Oxygraph软件的呼吸速率。 </li><li>进入正常化的因素:如果添加150微克的线粒体蛋白，正常化的因素将是0.15。 </li><li>规范化呼吸率计算出不同的呼吸状态。 </li><li>计算除以3国4国的呼吸控制率（RCR）。 </li></ol> 6。代表性的成果: <img src="/files/ftp_upload/2452/2452fig1.jpg" alt="图1" /> 图1显示了一个代表骨骼肌线粒体消耗氧气的跟踪。电极信号稳定后，线粒体样品添加到Oxygraph室。国家2呼吸开始除了与谷氨酸和苹果酸。此外ADP的增加氧的消耗，定义状态3呼吸。 ATP合酶阻断寡此外，获得国家4呼吸。最后，FCCP添加到解耦线粒体呼吸（状态5）， 表1显示了国2，3，4和5代表氧的消耗率。呼吸控制率（RCR）是计算每个实验。 RCR≥4被认为是一个可行的线粒体准备的证据。 <table border="1"><tbody><tr><td> 国家 </td><td> 归价格 </td></tr><tr><td>状态2 </td><td> 34.74 </td></tr><tr><td>状态3 </td><td> 153.40 </td></tr><tr><td>国家4 </td><td> 16.49 </td></tr><tr><td>国5 </td><td> 143.70 </td></tr><tr><td> RCR </td><td> 9.30 </td></tr></tbody></table> 表1。线粒体呼吸速率。Representativeoxygen骨骼肌线粒体的消费率。值进行归一室线粒体的添加量。呼吸控制率（RCR），是由分裂状态3状态4。 RCR≥4代表一个可行的线粒体准备。

<ol>
	<li>Frezza, C., Cipolat, S., Scorrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+isolation:+functional+mitochondria+from+mouse+liver,+muscle+and+cultured+fibroblasts.">Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts.</a> Nat Protoc. 2, 287-295 (2007).</li><li>Duchen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+mitochondria+in+health+and+disease.">Roles of mitochondria in health and disease.</a> Diabetes. 53, S96-S102 (2004).</li><li>Johannsen, D. L., Ravussin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mitochondria+in+health+and+disease.">The role of mitochondria in health and disease.</a> Curr Opin Pharmacol. , (2009).</li><li>Pallotti, F., Lenaz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+subfractionation+of+mitochondria+from+animal+cells+and+tissue+culture+lines.">Isolation and subfractionation of mitochondria from animal cells and tissue culture lines.</a> Methods Cell Biol. 80, 3-44 (2007).</li><li>Pallotti, F., Lenaz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+subfractionation+of+mitochondria+from+animal+cells+and+tissue+culture+lines.">Isolation and subfractionation of mitochondria from animal cells and tissue culture lines.</a> Methods Cell Biol. 65, 1-35 (2001).</li><li>Gamboa, J. L., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+content+and+distribution+changes+specific+to+mouse+diaphragm+after+chronic+normobaric+hypoxia.">Mitochondrial content and distribution changes specific to mouse diaphragm after chronic normobaric hypoxia.</a> Am J Physiol Regul Integr Comp Physiol. , (2009).</li><li>Patel, S. P., Gamboa, J. L., McMullen, C. A., Rabchevsky, A., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lower+respiratory+capacity+in+extraocular+muscle+mitochondria:+evidence+for+intrinsic+differences+in+mitochondrial+composition+and+function.">Lower respiratory capacity in extraocular muscle mitochondria: evidence for intrinsic differences in mitochondrial composition and function.</a> Invest Ophthalmol Vis Sci. 50, 180-186 (2009).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li></ol>

我们提出了一个协议孤立可行的骨骼肌线粒体。如果收益率是一个问题，该协议可以修改孵化5毫升30分钟，在冰PBS/10mM EDTA/0.01％胰蛋白酶的孤立肌肉。为了保证胰蛋白酶完成肌肉消化，肌肉需要充分剁碎。 PBS/10mM EDTA/0.01％胰蛋白酶溶液孵育30分钟后，必须完全取代隔离缓冲区1（IB1）3毫升。此外，使用胰蛋白酶，可能会干扰线粒体呼吸协议中的一些基板。这一协议处于良好的使用胰蛋白酶时线粒?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>95% CO2 / 5% O2 mix</td>
<td>&nbsp;</td>
<td>Local gas company</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine 5&#x2032;-diphosphate sodium salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A2754</td>
<td>&nbsp;</td>
<tr>
<td>Blue Rizla Paper</td>
<td>&nbsp;</td>
<td>Hansatech</td>
<td>890101</td>
<td>&nbsp;</td>
<tr>
<td>Bradford protein assay</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>500-0006</td>
<td>&nbsp;</td>
<tr>
<td>Carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2920</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge 5804R</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Compressed nitrogen</td>
<td>&nbsp;</td>
<td>Local gas company</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>D-mannitol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9647</td>
<td>&nbsp;</td>
<tr>
<td>Ethlyene-glycol-bis-tetraacetic acid (EGTA)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E3889</td>
<td>&nbsp;</td>
<tr>
<td>Ethylenediaminetetraacetic acid (EDTA)</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>161-0728</td>
<td>&nbsp;</td>
<tr>
<td>Free fatty acid bovine serum albumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A8806</td>
<td>&nbsp;</td>
<tr>
<td>Glutamic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G5889</td>
<td>&nbsp;</td>
<tr>
<td>HEPES sodium salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H7006</td>
<td>&nbsp;</td>
<tr>
<td>Isotemp 3006D</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M8266</td>
<td>&nbsp;</td>
<tr>
<td>Male Sprague Dawley Rats</td>
<td>&nbsp;</td>
<td>Harlan</td>
<td>300-500g</td>
<td>&nbsp;</td>
<tr>
<td>Malic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9138</td>
<td>&nbsp;</td>
<tr>
<td>Minifuge</td>
<td>&nbsp;</td>
<td>ISC Bioexpress</td>
<td>C1301P</td>
<td>&nbsp;</td>
<tr>
<td>Oligomycin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>O4876</td>
<td>&nbsp;</td>
<tr>
<td>Oxygen electrode disc</td>
<td>&nbsp;</td>
<td>Hansatech</td>
<td>S1</td>
<td>&nbsp;</td>
<tr>
<td>Oxygraph</td>
<td>&nbsp;</td>
<td>Hansatech</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Oxygraph Plus V1.01 Software</td>
<td>&nbsp;</td>
<td>Hansatech</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>pH-meter</td>
<td>&nbsp;</td>
<td>Mettler Toledo</td>
<td>1225506149</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate-buffered saline (PBS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4417</td>
<td>&nbsp;</td>
<tr>
<td>Potassium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P3911</td>
<td>&nbsp;</td>
<tr>
<td>Potassium phosphate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P8416</td>
<td>&nbsp;</td>
<tr>
<td>Potter-Elvehjem homogenizers</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>08-414-14A</td>
<td>&nbsp;</td>
<tr>
<td>PTFE (0.0125mm &#215; 25mm) membrane</td>
<td>&nbsp;</td>
<td>Hansatech</td>
<td>S4</td>
<td>&nbsp;</td>
<tr>
<td>SKIL 3320 drill press</td>
<td>&nbsp;</td>
<td>Hardware store</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S5016</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

mitochondrial isolation from skeletal muscle

线粒体是细胞器控制细胞死亡的生命和。他们参与重要的代谢反应，合成的ATP，规范的信号级联 2,3 。过去和目前研究人员已经从大鼠和小鼠的组织，如肝，脑和心脏4,5中分离出线粒体。近年来，许多研究人员都集中在研究从骨骼肌线粒体功能。 在这里，我们描述了，我们已经成功地用于从 6骨骼肌线粒体隔离的方法。我们的程序要求，所有的缓冲液和试剂都取得了新的和需要约250-500毫克骨骼肌。我们研究了从大鼠和小鼠腓肠肌和隔膜中分离出线粒体，鼠眼外肌。线粒体蛋白浓度测定Bradford法。重要的是保持在编制和功能的研究是一个相对较短的时间（约1小时）内进行线粒体样品冰冷。线粒体呼吸是用一个克拉克型电极（Oxygraph系统）在37 ° C 7极谱。氧电极的校准是在本议定书中的关键一步，它每天必须执行。隔离线粒体（150微克）被添加到实验缓冲液（EB）0.5毫升。国家2呼吸开始，除谷氨酸（5MM）和苹果酸（2.5毫米）。然后，二磷酸腺苷（ADP）（150微米）被添加到启动状态3。寡（1μM），ATP酶合成酶阻断剂，用于估计状态4。最后，羰基氰化物的P -三氟甲氧基] -苯基腙（FCCP，0.2微米）添加到measurestate 5，或耦合呼吸 6 。呼吸控制率（RCR），状态3状态4的比例，计算后，每一个实验。 RCR≥4被认为是一个可行的线粒体准备的证据。 总之，我们目前的隔离，可用于生化（例如，研究酶的活性，免疫检测，蛋白质组学）和功能（线粒体呼吸）的骨骼肌线粒体可行的方法。

Watch this Scientific Journal Video about Mitochondrial Isolation from Skeletal Muscle at JoVE.com

Mitochondrial Isolation from Skeletal Muscle

这个协议描述一个过程来研究从骨骼肌中分离出的线粒体呼吸。这种方法改编自Scorrano<em&gt;等。</em&gt;（2007年）。线粒体的分离过程中需要约2小时。线粒体呼吸在大约1个小时就可以完成。

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 ...

mitochondrial-isolation-from-skeletal-muscle

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

28.8K 次观看.  University of Kentucky. 这个协议描述一个过程来研究从骨骼肌中分离出的线粒体呼吸。这种方法改编自Scorrano等。（2007年）。线粒体的分离过程中需要约2小时。线粒体呼吸在大约1个小时就可以完成。

视频: Mitochondrial Isolation from Skeletal Muscle

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

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 ...

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.

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 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 Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

人体心室心肌细胞与颤音-切切心肌切片分离

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

从小鼠骨骼肌中分离线粒体用于呼吸测定

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in mice.