1。收集组织： <ol><li>使用7至12周龄的小鼠。通常情况下，至少有两种动物：一种是设立补偿和荧光减一的同型对照样品所需的流式细胞仪的单色控制。另一种是实际样品进行排序。 </li><li>安乐死小鼠CO 2吸入或根据选择的道德协议。放置纸巾，朝上，喷以70％的乙醇小鼠彻底防止污染用鼠标毛皮。 </li><li>提起腹部皮肤与钳，然后让一个小锋利的剪刀纵行剪开。 </li><li>剥离皮肤皮瓣在相反的方向（上下）完全暴露后肢肌肉下方。 </li><li>海关通过插入并延长剪刀沿胫骨两侧的肌肉组织。 </li><li>股骨重复同样的过程中提取的肌肉组织。 </li><li>放置在60毫米培养皿的菜全部切除的肌肉组织，组织从每个鼠标使用一盘。 </li><li>重复上述所有小鼠。 </li></ol> 2。酶消化分解成单细胞组织： 在本节的协议，使用消毒试剂和工具，并在无菌条件下工作。 <ol start="9"><li>第二胶原酶（Sigma公司，猫＃6885，500U /毫升）2毫升和20μL250MM氯化钙2股票添加到每个基于Petri盘。 </li><li>撕成小块（约1毫米3）注重用两个镊子（一锯齿形，与2牙齿，从精细的科学工具，猫＃11051-10，11154-10）取出并丢弃多少污染，肌肉组织尽可能的非肌肉组织。 </li><li>盖的Petri菜和孵育30分钟，摄氏37度。 </li><li>检索的Petri菜，并用3毫升注射器推杆彻底捣烂组织块。每个样品使用一个柱塞，以避免污染。 </li><li>添加一些（约5毫升）无菌冷PBS每个基于Petri盘，并转移到一个50毫升猎鹰管组织的污泥（如果要收回所有的组织，冲洗的Petri菜） </li><li>大力摇晃的Falcon管，填充管，用PBS离心@ 800rpm × 5分钟（Eppendorf公司的台式产品型号：5810R），吸出上清液（重复3次） </li><li>每管加入1毫升混合胶原酶ð（CAT＃11088882001，罗氏，1.5U / ML）/ Dispase II（罗氏，猫＃：04942078001，2.4u /毫升）和氯化钙210μL（250毫米），在37度孵育摄氏一小时轮换。 </li><li>添加5-10毫升冷，无菌PBS，旋涡和吸管彻底均质组织。 </li><li>填充管，用PBS，拌匀。 </li><li>转移到液体50毫升猎鹰管顶部放置一个40微米的细胞滤网过滤掉剩余的大块组织。每个样品匀成300毫升猎鹰管。填充每个管与PBS，然后在8摄氏度@ 1600rpm离心5分钟。 </li><li>弃上清，收集细胞进行染色。 </li></ol> 3。抗体染色和排序： 在本节的协议，使用无菌流式细胞仪和收集缓冲区，并在无菌条件下工作。商业抗体通常被认为是无菌的，如果处理得当。 <ol start="20"><li>单一的颜色和同型对照样品，重悬浮缓冲液1ml流式细胞仪的样品和除以900 1.5ml的预标记的Eppendorf管（100μL，每管）悬浮细胞。 <ol><li>设置单一颜色的染色混合根据如下表： 注意：在染色的同型对照抗体终浓度应符合他们控制的主要抗体 。 例如，如果使用反SCA - 1是1μg/106细胞，其相应的同型，必须使用在相同浓度。 <table border="1"><tbody><tr><td>管＃ </td><td>管名称</td><td>公司</td><td>猫＃ </td><td>克隆</td><td>同型</td><td>稀释</td></tr><tr><td> 1 </td><td>非染色</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 2 </td><td> Hoechst33342 </td><td>西格玛</td><td> B2261 </td><td></td><td></td><td> 2-5ug/ml </td></tr><tr><td> 3 </td><td>有价证券投资</td><td>西格玛</td><td> P4864 </td><td></td><td></td><td> 1ug/ml </td></tr><tr><td rowspan="2"> 4 </td><td rowspan="2"> CD31 - FITC CD45 - FITC </td><td> eBioscience公司</td><td> 11-0311-85 </td><td> 390 </td><td>鼠IgG2a </td><td> 500 </td></tr><tr><td>众议院提出</td><td></td><td> I3 / 2 </td><td>鼠IgG2b </td><td> 500 </td></tr><tr><td> 5 </td><td> SCA1 - PECY7 </td><td> eBioscience公司</td><td> 25-5981-82 </td><td> D7 </td><td>鼠IgG2a </td><td> 5000 </td></tr><tr><td> 6 </td><td> α- 7的APC </td><td>众议院提出</td><td></td><td> R2F2 </td><td>鼠IgG2b </td><td> 1000 </td></tr></tbody></table></li><li>设置同型对照染色混合使用下表： <table border="1"><tbody><tr><td>管＃ </td><td>名称 管</td><td>赫希斯特</td><td>有价证券投资</td><td> CD31 - FITC标记</td><td> CD45 - FITC标记</td><td> SCAL - PE - CY7 </td><td> A - 7 - APC的</td><td>鼠 IgG2b - APC的</td><td>鼠 IgG2a - pecy7 </td><td>鼠 IgG2a - FITC标记</td><td>鼠 IgG2b - FITC标记</td></tr><tr><td> 7 </td><td> ISO - CD31 / CD45 </td><td> + </td><td> + </td><td> - </td><td> - </td><td> + </td><td> + </td><td> - </td><td> - </td><td> + </td><td> + </td></tr><tr><td> 8 </td><td> ISO - SCAL - pecy7 </td><td> + </td><td> + </td><td> + </td><td> + </td><td> - </td><td> + </td><td> - </td><td> + </td><td> - </td><td> - </td></tr><tr><td> 9 </td><td> ISO - α- 7 - APC </td><td> + </td><td> + </td><td> + </td><td> + </td><td> + </td><td> - </td><td> + </td><td> - </td><td> - </td><td> - </td></tr></tbody></table></li><li>从第20步到适当的Eppendorf管吸取单一的颜色和同位素控制染色混合物（我们通常调整的抗体浓度，使5至10μL染色混合添加到每个样品）。充分混合，并在冰上孵育1小时。 </li></ol></li><li>从样品鼠标细胞染色抗体鸡尾酒800μL流式细胞仪细胞分选（下图）。充分混合，并在冰上孵育1小时。 抗体鸡尾酒：赫司特，PI，CD31 - FITC，CD45 - FITC标记，PECY7 SCA1 -α- 7 APC 注意：对细胞抗体的最佳比例必须由单独滴定每个抗体，作为批次之间可能存在显着差异决定的 。 在每10 6靶细胞抗体μgs和抗体的最佳的使用量应表示使用同一批次的抗体时，保持不变。 显然，如果不靶细胞的数量显着变化之间的实验，或者，如果染色卷细胞的数量与规模，每个抗体的固定稀释也可以使用。 </li><li>洗：流式细胞缓冲9对照管各加入约1毫升;增加约流式细胞仪缓冲液20ml样品管。 </li><li>离心5分钟Eppendorf管@ 3000RPM（Eppendorf公司台式离心机，型号：5417C）。离心机样品管@ 1600rpm 5分钟（Eppendorf公司台式离心机，型号：5810R）。 </li><li>吸弃管上清。 </li><li>悬浮细胞，在流式细胞仪缓冲区（1ML每个控件，排序样本4ML）移液器细胞进入“流式细胞仪管”通过细胞过滤器盖（屋宇署猎鹰猫＃352235，孔径40微米），去除剩余的团块可能影响的流式细胞仪。 </li><li>成立单一的色彩控制和同型对照流式细胞仪分选机。 </li><li>根据下表（95％DMEM培养液，5％FBS）分别在3.5毫升收集缓冲区收集排序细胞。通常情况下，收获两个后肢时，一个单一的野生型小鼠可产生大约15万生肌祖细胞或100000成脂分化的祖细胞，95％以上的可行性，用流式细胞仪来判断在该过程结束时重新分析排序细胞（图1 ） 表面染色的基础上生肌和成脂分化的祖细胞的定义 <table border="1"><tbody><tr><td>抗体/染色</td><td>生肌祖（MP） </td><td>成脂祖（AP） </td></tr><tr><td>赫希斯特</td><td> + </td><td> + </td></tr><tr><td>有价证券投资</td><td> - </td><td> - </td></tr><tr><td> CD31 - FITC </td><td> - </td><td> - </td></tr><tr><td> CD45 - FITC </td><td&gt; - </td><td> - </td></tr><tr><td> SCA1 - PECY7 </td><td> - </td><td> + </td></tr><tr><td> α- 7的APC </td><td> + </td><td> - </td></tr></tbody></table></li></ol> 4。移植： <ol start="28"><li>离心机排序细胞@ 1500RPM 5分钟，取出上清液和转让细胞灭菌Eppendorf管（1.5毫升），500μL无菌PBS（无血清！）冲洗细胞，然后离心5分钟@ 3000RPM。所有的试剂，并在无菌条件下保持在一个组织的文化罩工作，重悬在约10 6细胞/ mL PBS中，或在基质胶成脂祖（屋宇署猫＃354234）生肌祖。 </li><li>移植生肌的祖细胞或成脂祖受体小鼠。 <ol><li> isoflouran根据机构的政策与麻醉鼠标。 </li><li>剃须注射区周围的头发，然后消毒，用70％乙醇擦的皮肤</li><li>生肌祖细胞或成脂分化的祖细胞（= 20K细胞），使用3 / 10的CC胰岛素注射器注入20μL（屋宇署猫＃309300）。 </li></ol></li><li>经过适当的时间（三个星期，但表达捐助衍生的转基因标记的肌纤维通常在移植后1周明显），麻醉受体小鼠腹腔注射400μL，Avertin（西格玛猫＃T04840 - 2从步骤29， 25毫克/毫升），再灌注transcardially毫升PBS（含EDTA的10微米）第一15ML，4％的煤灰。收集与4％煤灰一夜之间的目标组织，修复后，如果需要的话，然后转移到20％蔗糖过夜。嵌入在华侨城，冻结和cryosection。 </li></ol> 5。代表性的成果： 当MP移植到小鼠骨骼肌，他们爽快地保险丝与预先存在的肌纤维。因此，任何在供体细胞的遗传标签，将在收到他们的纤维，很容易检测到。图2显示了一个例子移植的细胞中表达人碱性phostphatase，显示组织化学。 AP都移植的结果是高度依赖移植部位的环境：当皮下注射移植这些细胞起源于脂肪细胞和肌纤维母细胞（见图2）。在许多其他网站，包括肌肉，他们无法生存，除非已经脂肪变性引起的3。 <img src="/files/ftp_upload/2476/2476fig1.jpg" alt="图1" /> 图1。骨骼肌祖种群隔离的排序策略 （一）排序策略 ：基于前向散射和侧向散射活菌。 Hoechst染色是用来排除anuclear碎片和碘化丙啶（PI）染色排除死细胞。排序盖茨造血（CD45）和血管内皮细胞（CD31）被排除在外。在α7+赫斯特+ PI - CD45 - CD31 - SCAL -子集包含所有祖细胞（MP），生肌。 SCAL +赫斯特+ PI - CD45 - CD31 - α7 -人口包含祖（AP）成脂。 （二）荧光减一（FMO）的同型对照确认污渍的特殊性。 （三）纯度检查排序后的MP和AP亚群。 <img src="/files/ftp_upload/2476/2476fig2.jpg" alt="图2" /> 图2。 MP和AP移植。美联社代表结果皮下移植（顶部面板）和MP肌肉注射（底部面板）。在这两种情况下，供体细胞的起源从表达的转基因人类碱性磷酸酶的老鼠，棕黄色染色确定。

<ol>
	<li>Buckingham, M., Montarras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+stem+cells.">Skeletal muscle stem cells.</a> Curr Opin Genet Dev. 18, 330-336 (2008).</li><li>Relaix, F., Marcelle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+stem+cells.">Muscle stem cells.</a> Curr Opin Cell Biol. 21, 748-753 (2009).</li><li>Joe, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+injury+activates+resident+fibro/adipogenic+progenitors+that+facilitate+myogenesis.">Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis.</a> Nat Cell Biol. 12, 153-163 (2010).</li><li>Natarajan, A., Lemos, D. R., Rossi, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibro/adipogenic+progenitors:+A+double-edged+sword+in+skeletal+muscle+regeneration.">Fibro/adipogenic progenitors: A double-edged sword in skeletal muscle regeneration.</a> Cell Cycle. 9, (2010).</li><li>Sherwood, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+adult+mouse+myogenic+progenitors:+functional+heterogeneity+of+cells+within+and+engrafting+skeletal+muscle.">Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle.</a> Cell. 119, 543-554 (2004).</li><li>Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+and+expansion+of+single+transplanted+muscle+stem+cells.">Self-renewal and expansion of single transplanted muscle stem cells.</a> Nature. , (2008).</li><li>Montarras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+isolation+of+satellite+cells+for+skeletal+muscle+regeneration.">Direct isolation of satellite cells for skeletal muscle regeneration.</a> Science. 309, 2064-2067 (2005).</li></ol>

本议定书的目标是取得一个合理的平衡之间的高收益和高纯化的细胞活力。可以预见，在最关键的一步，确保健康的细胞恢复，开始组织酶解。组织的处理应特别温柔，这是很难证明或欣赏视听格式甚至的。另一个关键因素是程序的长度。时间越长，去捐助给收件人动物的可行性较低，因此，植入的效率。如果有任何不立即通过PI在纯化后的细胞样本阳性事件排序后的频率回答关于可行性的问题，我们建议，是一个限制的稀释检测，以测量clonogenicity 3。各种典型完好的肌肉经常在15-20细胞能够在体外生肌或纤维/成脂殖民地发起产量约1。虽然从国会议员发起的殖民地，基本上100％包含有区别的肌管，从FAPs获得的殖民地只有约三分之一包含除了平滑肌肌动蛋白的积极肌纤维母细胞中的脂肪细胞。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>胶原酶二</td><td> Sigma - Aldrich公司</td><td> C - 6885， </td><td> 500 U / ml的</td></tr><tr><td>胶原酶ð </td><td>罗氏公司</td><td> 11088882001 </td><td> 1.5 U / ml的</td></tr><tr><td>置换第二</td><td>罗氏公司</td><td> 04942078001 </td><td> 2.4 U /毫升</td></tr><tr><td>细胞过滤器</td><td>屋宇署</td><td> 352340 </td><td> 40微米</td></tr><tr><td>管细胞过滤器</td><td>屋宇署</td><td> 352235 </td><td> 40微米</td></tr><tr><td> Hoechst33342 </td><td> Sigma - Aldrich公司</td><td> B2261 </td><td> 2-5微克/毫升</td></tr><tr><td> CD31 - FITC </td><td> Ebiosciences </td><td> 11-0311-85 </td><td>克隆390 </td></tr><tr><td> CD45 - FITC </td><td>自制</td><td></td><td>克隆I3 / 2 </td></tr><tr><td> SCA1 - PECY7 </td><td> Ebiosciences </td><td> 25-5981-82 </td><td>克隆的D7 </td></tr><tr><td> α- 7整合APC </td><td>自制</td><td>供应实验室与罗西</td><td> R2F2 </td></tr></tbody></table>

purification of progenitors from skeletal muscle

骨骼肌包含多个不同的胚胎起源和发展潜力的祖人口。生肌祖细胞，通常居住在“卫星细胞的位置”之间的肌纤维质膜和层粘连蛋白丰富的基底膜，ensheaths，自我更新的细胞仅致力于生肌血统 1,2 。最近，我们所描述的船只相关的祖细胞，能够产生肌纤维母细胞和白色脂肪细胞，这是为了响应有效地进入扩散损害和肌细胞提供营养支持在组织再生3,4第二类。其中一个最值得信赖的分析来确定一个特定的细胞群的发育和再生的潜力依赖于他们的分离和移植5-7。为此，我们已经优化了其净化的协议，通过流式细胞仪，酶分离肌肉，我们将在这篇文章中概述。使用这种方法得出的人口将包含生肌或纤维/成脂细胞集落形成细胞，没有其他类型的细胞，能够扩大在体外或体内交货尚存。然而，当这些人群分子之一必须牢记，新鲜排序的子集可能包含其他污染物细胞缺乏形成的殖民地或嫁接受助人的能力的分析（如QRT - PCR）排序后立即使用。

Watch this Scientific Journal Video about Purification of Progenitors from Skeletal Muscle at JoVE.com

Purification of Progenitors from Skeletal Muscle

酶解，表面标记和流式细胞仪检测纤维/成脂和小鼠骨骼肌生肌祖净化的方法。

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 ...

purification-of-progenitors-from-skeletal-muscle

Research

JoVE Journal

Biology

18.1K 次观看.  University of British Columbia. 酶解，表面标记和流式细胞仪检测纤维/成脂和小鼠骨骼肌生肌祖净化的方法。

视频: Purification of Progenitors 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

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 ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营：从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

激光伤害的斑马鱼胚胎骨骼肌

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

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 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 ...

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

大鼠骨骼肌中纤维-脂肪源性祖细胞（FAPs）和肌源性祖细胞（MPs）的鉴定、分离和表征

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in ...

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 ...