我们想感谢萨拉·迪克提供批判性阅读和评论。 MAR持有加拿大研究主席，分子遗传学和霍华德休斯医学研究所是一个国际研究学者。这项工作得到了国家卫生研究院，霍华德休斯医学研究所，加拿大卫生研究院的研究，肌肉萎缩症协会和加拿大研究主席计划拨款，MAR。

所有的实验都根据动物照顾和处理的大学渥太华规定的处理。 请参阅表1概述了协议。 1。开始之前的分离<ol><li>准备以下解决方案： 0.2％胶原酶I型在DMEM（Dulbecco改良的Eagle氏培养基，高葡萄糖，L-谷氨酰胺，110毫克/毫升丙酮酸钠）。对于两个EDL肌肉，准备在DMEM 0.2％Collagenease 2毫升。通过0.22微米的过滤器，过滤器的解决方案。 10分钟前隔离，prewarm在37℃下在水浴中。其他等份的未稀释的胶原酶，可以在-20℃下冷冻，以供以后使用。 注意：与丙酮酸钠整个所有的过程中使用的DMEM。纤维不生存在丙酮酸钠无介质。 <ul><li>清洗介质（用来执行所有的清洗）。补编用1％青霉素/链霉素的DMEM。通过过滤器0使用0.22微米的过滤器之前。 </li><li>肌纤维的培养基。补编的DMEM，20％FBS，1％鸡胚胎提取物和1％青霉素/链霉素。通过0.22微米的过滤器在使用前过滤。 </li><li>基质胶涂层的菜肴。文化纤维很长一段时间，我们建议使用基质胶涂层基体。为了准备基底膜的涂覆菜肴，解冻的Matrigel的等分试样，在4℃下过夜。后的第二天，1:10稀释Matrigel的DMEM。查看基底膜，在4℃下在所有时间，避免突然的温度变化，因为这将Matrigel的解决方案，从而导致不​​均匀的涂层内创建的微小晶体。地点菜肴被涂覆在冰上。外套菜具有足够的容量以覆盖面。让它坐1分钟。完全移除剩余的基底膜。让菜在37°C干燥，使用前至少3小时或过夜。 </li></ul> 注意：等分试样稀释的基底膜可以重新使用多次用于涂覆目的如果4和de存储克; C. </li><li>最后，用70％的乙醇，一定要清洁显微镜站和解剖工具。 </li><li>对于2的EDL隔离（一个鼠标）准备5个塑料培养皿（60 * 15MM）如下： <ul><li>四道菜的隔离（1为肌肉分解，3的串行洗涤）。所有菜肴必须涂上马血清（HS），以防止肌纤维附于塑料。外套，移液管3毫升到每道菜马血清，漩涡，使涂层均匀，取出马血清和至少30分钟，让菜干。加入4毫升的DMEM，每道菜。查看菜在37℃下，在5％CO 2培养箱中，在使用之前。 </li></ul> 注：马血清可以重新使用多次，如果保持无菌。可替换地，可以使用10％的溶液，HS在DMEM涂覆菜肴。使用HS涂在整个协议的菜肴和培养肌纤维浮动条件。 <ul><li>一个菜将被隔离后用于培养肌纤维。 Alt键取决于下游应用的最终培养的肌纤维可用于ernative菜的尺寸或格式。但是，我们不建议盘尺寸大于60×15毫米。肌纤维可以保持停牌时间不超过96小时之前发生超收缩。对于更长的培养时间，我们建议使用的基质胶涂层的菜肴或板。 </li></ul></li><li>对于一个光纤隔离（EDL），准备两个无菌的巴斯德移液管：大口径吸管为的肌肉处理和一个小口径的吸管肌纤维操纵。使用金​​刚石笔削减各玻璃移液管到所需的长度和热抛光移液器的边缘平滑。通过使用火焰，曲线的前端的小口径的吸移管。这将有助于处理单纤维。火焰消毒。 HS使用前，每个移液器外套。 </li></ol> 2。肌肉解剖和消化<ol><li>这些实验的目的，8周龄SV129，PAX7 CreER Cklr; Rosa26TdTomato（被用来Pax7Cre-TdTomato） 和Myf5-Cre重组酶; Rosa26YFP。 </li><li>喷用70％乙醇的后肢。脚的动物（正面朝上），支撑板的后肢在操作过程中有更好的把握。 </li><li>随着剪刀的帮助下，切断通过肢体的整个长度上，露出下面的肌肉。去除皮肤以及任何毛发或毛皮（ 图1A）。 </li><li>用细剪刀，切透薄的筋膜，不破坏下面的肌肉。视觉定位EDL。 EDL前室正下方的后肢胫前肌（TA）肌肉中发现。 </li><li>随着两钳的帮助下，暴露的远端肌腱。 </li><li>切断远端肌腱（TA和EDL）与的尖锐Cohann-Vannas春风似剪刀。 </li><li>借助产钳举行TA和他们的肌腱和微妙的EDL肌肉拉向近端的肌肉。在这一点上，你应该能够清楚地看到EDL肌止点下方的TA肌肉。现在，从TA肌肉分离的EDL，在相反的方向（ 图1B）通过拉动两筋。避免在执行此操作，因为这将损坏的肌纤维拉伸EDL肌肉。 </li><li>公开EDL肌腱。为了更好地可视化近端肌腱在这一点上，它可以帮助删除的TA肌肉。它也可能有助于减少一些膝关节周围的结缔组织（ 图1C）。 </li><li>把近端肌腱，轻轻取出的EDL（ 图1D）。 </li><li>通过保持通过其肌腱的肌肉，把它传输到2毫升预先制备的胶原酶溶液。在水浴中，在37℃下孵育。 注：成功的纤维隔离，重要的是要隔离的EDL使肌腱到肌腱的肌纤维的完整性保持。在解剖显微镜下，可以执行步骤2.3到2.9。可替代地，使用一个MAGNifying玻璃也可能有助于肌肉有更好的意见。 </li><li>重复步骤2.3至2.9隔离第二EDL。转移在同一试管中的第二的EDL。为了避免不均匀的消化，分离所述第二的EDL应不超过5分钟，完成后的第一个。 注1：培养时间取决于胶原酶活性，可能需要进行调整。或长或短的潜伏期可能需要根据不同的大小，年龄和/或肌肉条件（ 例如肝纤维化的肌肉需要较长的消化时间）。 注2：如果检查卫星细胞在静止条件下的行为，情绪激动可以激活卫星细胞在肌肉消化时间10。 </li><li>在消化时间内，定期检查，以避免肌肉过度消化。当肌肉开始放松，肌纤维是可见的，停止消化。要停止消化，仔细肌肉同时传送到一个​​预热的培养皿中，用4毫升的DMEM（dissociati的上盘）。使用大口径的大小吸管来执行此操作。 注意事项：避免肌肉过量，因为这不可避免地导致超收缩肌纤维的隔离。 </li></ol> 3。单肌纤维的游离与培养<ol><li>要释放的肌纤维，使用大口径的玻璃吸管，用温水中，直到冲洗肌肉纤维自然开始被释放。不要捣碎的肌肉，因为这将不可避免地造成破坏的纤维。请执行以下步骤在解剖显微镜下。 </li><li>继续释放，直到达到所需的号码的肌纤维。如果在室温下为10分钟以上的菜需要允许5 min（最小值）的温育于37℃下，5％CO 2的重新平衡的介质。 注：如果介质到达一个较长的时间低于生理温度（37°C），肌纤维就会死亡。 </li><li>使用小尺寸的孔移液管，内部转移ŕ住单肌纤维到一个新的的预热盘（连续三洗一）。而不是把大量的肌纤维在一次单独处理每个肌纤维。如果有必要，孵育在37℃，5％CO 2的10-15分钟，以重新平衡介质。 </li><li> 3.3 2次以上重复步骤，直到所有的死肌纤维和杂物被删除。我们建议至少3个连续的清洗进行适当的清理。 注：死肌纤维出现的短期和超签约，在显微镜下光。 </li><li>孵育单肌纤维在37℃，5％CO 2中的至少一个小时之前，切换到培养基培养皿（DMEM中只）在最后一次洗涤。这允许调整到的肌纤维中的无血清在体外条件下。我们发现，立即文化的肌纤维，提高纤维萎缩的血清营养丰富的培养基。 </li><li> 1小时后，转移到一个新的预热盘的肌纤维或适当的文化形式取决于下游应用。高血清培养基的文化纤维，使卫星细胞活化。或者，不同浓度的血清或鸡胚提取物也可以被使用。切换介质隔日。 </li></ol> 4。下游的应用<ol><li> 免疫组织化学染色 。供应信息肌纤维可以在任何时间点上固定和染色过程中隔离。免疫荧光法，可以进行上漂浮和基板连接的肌纤维。 ，如果染色浮动的肌纤维，用一个小口径的玻璃吸管转移肌纤维从一个解决方案。或者，它可以保持在相同的孔中的肌纤维在整个所有的程序和添加/删除解决方案，使用的玻璃移液管。避免标准，因为这将导致肌纤维，以及去除的愿望的。简要地说，完全除去培养基，预热5分钟的4％多聚甲醛（PFA）的修复肌纤维。广泛洗在PBS几次。温浴，肌纤维1％甘氨酸的PBS或其他规范淬火解决方案，以最大限度地减少，PFA背景染色。如果有必要，透性肌纤维与0.1％的Triton X-100的PBS和10分钟，然后在PBS中洗涤5分钟。孵育1小时，在室温下或优选在4℃下过夜封闭液（10％马血清，0.1％的Triton X-100，1％NaN）的纤维可以使用替代封闭液，取决于感兴趣的抗体。在PBS中洗涤一次5分钟。与适当的主用封闭液稀释，在室温下1小时或过夜的抗体在4℃下孵育洗纤维在PBS洗5分钟，每3次，以除去未结合的抗体。适当的二次抗体，在室温下45分钟至1小时的孵育。在PBS中洗涤5分钟，每3次洗净。染液，用1 mg / ml的DAPI核。 ，如果浮游纤维染色，将每个光纤适用于显微镜载玻片上。删除任何多余的PBS或中等。应用安装的Medi嗯，然后加盖玻片。在荧光显微镜下进行可视化的肌纤维。请参阅图4为代表的免疫EDL肌纤维。使用更强的固定剂的替代染色程序描述维尔马，M. 等人，11和Wosniak，AC 等12。 </li><li> 寡核苷酸或质粒瞬时转染 。活肌纤维可以转染质粒或针对特定基因的siRNA / s的兴趣。利用siRNA时，双转染高效的基因敲除的建议。我们建议进行转染后8小时的隔离。六小时后，转染，用新鲜培养基替换。对于siRNA的转染，我们建议通过使用最终浓度为50nM开始。 RNA的提取，或优选地通过免疫染色，可以分析基因表达击倒。 </li><li> 病毒感染 。感染的活肌纤维细胞的发病率是可能的，虽然死亡是高于与寡核苷酸的转染和感染的效率可以是可变的，取决于肌肉条件和年龄。例如，完整的成人肌肉的肌纤维特别是非响应于病毒感染，由于这已被证明，以提供保护屏障，防止宿主感染13，14的存在下的基底层。是优选的慢病毒载体，逆转录病毒载体，因为它们可以感染静态（非有丝分裂）细胞。病毒感染，最好是一个Matrigel的涂层盘或板和肌纤维调节介质条件下的第一个24小时的培养纤维。 </li><li> 实时成像 。实时成像的肌纤维特别是耗时的，并必须在37℃，5％CO 2的腔室的显微镜配备。它是有用的，当评估单颗卫星细胞的行为。使用这种技术的工作一直卫星细胞的异质性的发现和研究他们的BEHAvior纤维（15和16）。 </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pax7+is+required+for+the+specification+of+myogenic+satellite+cells.">Pax7 is required for the specification of myogenic satellite cells.</a> Cell. 102 (6), 777-786 (2000).</li><li>Zammit, P., Golding, J. P., Nagata, Y., Hudon, V., Partridge, T. A., Beauchamp, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+satellite+cells+adopt+divergent+fates:+a+mechanism+for+self-renewal.">Muscle satellite cells adopt divergent fates: a mechanism for self-renewal.</a> J. Cell Biol. 166 (3), 347-357 (2004).</li></ol>

没有利益冲突。

单从完整的肌肉的肌纤维的分离和培养提供了一个极好的体外模型研究的过程中，肌肉再生。该系统的一个独特的功能是保存的卫星细胞的生理环境下的基底层。最重要的是，该技术可用于研究在两个静止和活化的状态的肌肉干细胞的行为。在过去的20年中，肌纤维培养系统提供了有意义的见解，卫星细胞的生物学人口与内在和外在的决定因素。通过培养个人的肌纤维，卫星细胞的异质性肌纤维，肌肉型或再生的潜力已经解决15。精心研究使用的单一培养的纤维允许卫星细胞迁移模式16获得信息的实时成像。的定量数据的采集是一个莱索托。虽然费时的，它提供了显着的分布和发生特定事件（干细胞不对称分裂，增殖和分化比等）的信息。与先前描述的应用程序一起，从单纤维是蛋白质或RNA的提取也可以，虽然隔离通过FACS或其他装置的卫星细胞的纯人口调查蛋白质或基因的表达，在分子水平上的变化，可能提供一个更好的平台。根据我们的经验，成功的光纤隔离最关键的一步是“筋筋”隔离（在协议文本的步骤2.5至2.9）。这可以保证，肌肉消化后，纤维被释放与最小或没有损害，从而提高它们的性能在下面的步骤从EDL。

<table border="1"><tbody><tr><td></td><td></td><td></td><td> 试剂/材料 </td></tr><tr><td>胶原酶</td><td> Sigma-Aldrich公司</td><td> C0130-1G </td><td>准备新鲜的所有时间，以获得最佳效果。于-20℃下可以存储其他等份的未稀释的胶原酶</td></tr><tr><td> DMEM高糖+ NaPyr </td><td> Invitrogen公司</td><td> 11995073 </td><td>此外，青/链霉素是必要的，以减少空气污染</td></tr><tr><td>特点胎牛血清</td><td> Hyclone公司</td><td> SH30396.03 </td><td>第＃KUJ35152地块</td></tr><tr><td>马血清</td><td> Hyclone公司</td><td> SH300.74.03 </td><td>第＃AVJ82494地块</td></tr><tr><td>鸡胚提取物</td><td>精确的化学</td><td> CE-650-T，批量B7035010 </td><td>避免冻融的周期。 </td></tr><tr><td>基底膜</td><td> BD </td><td></td><td>将等分试样储存于-20℃下避免冻融的周期。将稀释后分装4°C </td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td> Cohann Vannas春季的剪刀脚6毫米刀片直夏普</td><td>精细科学工具</td><td> 15000-02 </td><td></td></tr><tr><td>超薄虹膜剪10.5厘米</td><td>精细科学工具</td><td> 14088-10 </td><td></td></tr><tr><td>莫里亚光圈钳弯</td><td>精细科学工具</td><td> 11373-12 </td><td></td></tr><tr><td>巴斯德吸液管</td><td> VWR </td><td> 14672-380 </td><td> 14672-200 </td></tr><tr><td>金刚石笔</td><td> VWR </td><td> 52865-005 </td><td></td></tr><tr><td> 60 * 15MM培养皿</td><td> VWR </td><td> 25384-092 </td><td></td></tr><tr><td>的Acrodisc 0.22微米过滤器</TD&gt; <td> VWR </td><td> CA28-145-477 </td><td></td></tr><tr><td>解剖显微镜StemiV6 </td><td>蔡司</td><td></td><td>可能需要额外的光源有更好的重点查看</td></tr></tbody></table>

isolation and culture of individual myofibers and their satellite cells from adult skeletal muscle

肌肉再生的成人进行的居民称为卫星细胞的干细胞。卫星细胞被定义为他们的基底层之间的位置和每个肌纤维的肌膜。目前的知识，他们的行为在很大程度上依赖于使用的单个肌纤维隔离协议。比肖夫于1985年，描述了一个协议，来隔离从成年大鼠趾短屈肌（FDB）的目标是建立在体外系统中，肌纤维和其干细胞之间的物理缔合被保留1单活纤维。 1995年，Rosenblattmodified比肖夫协议单独挑选和处理，使肌纤维分开后，胶原酶消化，而 ​​不是孤立的重力沉降2，3。罗森布拉特或比肖夫协议已经被调整到不同的肌肉，年龄或条件3-6。单肌纤维隔离技术是不可缺少的工具，由于其独特的优势。首先，在单肌纤维协议，卫星细胞保持下面的基底层。这是一个独特的功能，协议，其他如荧光激活细胞分选技术要求化学和机械组织分离。虽然肌纤维的文化系统无法代替的体内研究，它提供了一个很好的平台，以解决有关的肌肉干细胞的生物学特性。单肌纤维的培养可以在标准的镀敷条件下或在浮动条件。卫星细胞上浮动肌纤维的比肌纤维的环境，几乎没有其他影响。基材的刚度和涂层已被证明影响卫星细胞的能力，再生肌肉8，9，以便能够独立地控制每个这些因素允许利基依赖性和独立反应的区分。不同浓度的血清也被证明有效果上从静止过渡激活。为了保持其相关联的卫星细胞的静止状态，纤维应保持在低血清培养基中1-3。研究基因在静止状态时，这是特别有用的。在血清营养丰富的培养基中，卫星细胞迅速激活，增殖，迁移和分化，从而模拟体内再生过程中的 1-3。该系统可用于执行各种的检测，如测试的化学抑制剂;提供由病毒的基因的异位表达;寡核苷酸为基础的基因敲除或实时成像。此视频文章介绍了目前使用的协议，在我们的实验室分离出单个肌纤维的趾长伸肌（EDL）肌肉成年小鼠（6-8周龄）。

在这里，我们描述了从成年小鼠的EDL肌肉孤立的单个肌纤维。成功的肌纤维产量取决于几个因素，如胶原酶的活性或肌肉疾病或年龄。最重要的是，隔离的肌肉由肌腱到肌腱的查询结果在长，完整的EDL的肌肉的肌纤维从图1示出步骤的图形表示“肌腱到肌腱”隔离的步骤。肌肉一旦被完全消化，肌纤维被释放通过温和的压力施加到肌肉的肌纤维隔离的第一次洗涤后的实验， 图2A示出了具有代表性的图象。在这一点上，文化包含的混合物长，闪亮的管状结构出现单个肌纤维束，过度收缩肌纤维，这是黑暗的，短期及碎片的消化过程。由端部的至少三个连续的洗涤液中，只有单活肌纤维应该留在二sh为文化或下游分析（ 图2B），图2C示出了长，现场的光纤作为一个超收缩的纤维（C&#39;）相比。在隔离的时间，卫星细胞出现微小的突起上的肌纤维的表面（ 图3A）。如果维持血清营养丰富的培养基中，卫星细胞的激活，增殖，迁移和最终融合成肌管（B）。经过15天的培养，所有的肌管表达MYF5记者的YFP（C），在成人的肌肉再生的细胞，在某些时候他们的发展过程中曾表示，生肌的决定因素MYF5的。所有的卫星细胞表达的配对box转录因子PAX7 17。记者的鼠标线Pax7Cre-TdTomato可以用于跟踪卫星细胞通过的TdTomato荧光信号（ 图3D和 E）中的表达， 图4示出了一个有代表性的免疫nofluorescence的PAX7 + / MYOD的双卫星细胞。经典的双PAX7 + / MYOD +卫星细胞被认为是细胞增殖的承诺将完成的肌肉祖细胞的分化程序通过下调PAX7 MyoD的表达，同时保持或恢复到静止通过下调MyoD和维护PAX7表达18 <img src="/files/ftp_upload/50074/50074fig1.jpg" alt="图1"/> 图1。 EDL从小鼠后肢肌肉隔离。一个 8周龄成年小鼠后肢的。箭头表示肌腱的远端（膝盖）和近端（脚）肌腱B。 （TA）的胫骨前肌和趾长伸肌（EDL）肌肉。C的解剖位置。通过保持通过近侧肌腱的EDL，肌肉被拉向膝盖露出的前端的肌腱。D。远端肌腱削减和的EDL释放。 <img src="/files/ftp_upload/50074/50074fig2.jpg" alt="图2"/> 图2。一个单一的肌纤维隔离实验的代表性结果。 A。亮场图像的肌纤维分离实验在第一次清洗步骤。红色箭头表示活肌纤维，白箭头表示超合同肌纤维和黄色箭头表示细胞，肌纤维或ECM碎片B。经过连续的DMEM清洗，只能活肌纤维仍然存在。C和C&#39;。图片是一个长期的完整的活肌纤维（C）和一个短的过度收缩肌纤维（C&#39;）。酒吧10微米。用Zeiss Axio上观察Z1 AxioCam HR配备显微镜照片拍摄。 <img src="/files/ftp_upload/50074/50074fig3.jpg" alt="图3"/> FIGUR<html> E 3。代表一个单一的肌纤维培养实验的结果。甲。布赖特场图像中的一个单一的，活的肌纤维与其相关的卫星细胞（箭头），右后的隔离（时间0），B。单肌纤维镀基底膜和血清富培养基中培养15天。白箭头指示分化的肌管相比，单细胞（黄色箭头）C。单肌纤维从Myf5Cre; RosaYFP鼠标作为B 中培养。箭头指示MYF5衍生的肌管。D和 E。单从Pax7Cre; TdTomato肌纤维分离，固定的权利。箭头表示PAX7积极静止卫星细胞（E，红色）。核复染用DAPI（D）。酒吧：50微米。用Zeiss Axio上观察Z1 AxioCam HR配备显微镜照片拍摄。 办法“&gt; <img src="/files/ftp_upload/50074/50074fig4.jpg" alt="图4"/> 图4。单肌纤维的肌纤维卫星细胞免疫荧光染色的例子。进行分离和培养72小时浮动条件。卫星细胞的肌纤维进行染色卫星细胞的特异性标记物PAX7（B，红色）的生肌调节因子MYOD的（C，绿色）。核复染用DAPI（A）。酒吧：50微米。用Zeiss Axio上观察Z1 AxioCam HR配备显微镜照片拍摄。 <table border="1" fo:keep-together.within-page="always"><tbody><tr><td> 肌肉解剖 （5分钟每一块肌肉） </td><td><ul><li>肌腱肌腱隔离的</li><li>工欲善其事，必先</li><li>最小的肌肉损伤</li></ul></td></tr><tr><td> 肌肉消化 （30-45分钟） </td><td><ul><li>温度敏感</li><li>避免过量</li></ul></td></tr><tr><td> 光纤隔离 </td><td><ul><li>避免过度操纵的纤维</li><li>多次洗涤消除碎片</li></ul></td></tr><tr><td> 光纤文化 （3-4周） </td><td><ul><li>格式：任何</li><li>涂层：马血清，基底膜</li><li>中：基底或高血</li></ul></td></tr></tbody></table> 表1中。概述的肌纤维肌纤维隔离协议。的隔离协议包括4个主要步骤。对于每个步骤的大致时间和主要的关键点进行了讨论。对于每个步骤的详细讨论，请参阅协议文本。

Watch this Scientific Journal Video about Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle at JoVE.com

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

肌纤维的分离和培养是黄金标准<em在体外</em系统研究通过平静，激活和分化的过渡卫星细胞。重要的是，单个肌纤维的文化系统保留的肌纤维/干细胞协会，这是一个必不可少的组成部分的肌肉干细胞小生境。

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 ...

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Biology

62.1K 次观看.  Ottawa Hospital Research Institute. 肌纤维的分离和培养是黄金标准

视频: Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult 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 ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

成人从人体皮肤的上皮干细胞的分离和培养

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

从解离分析准备胚胎和幼体斑马鱼骨肌纤维

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

分离，培养和移植肌卫星细胞

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

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 and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

DNA电穿孔，肌纤维的分离和成像

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

来自新生儿和成年鼠皮肤的原代小鼠角化细胞的分离和培养

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

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 ...