Name: in situ immunofluorescent staining of autophagy in muscle stem cells
Uploaded: 2017-06-12T15:00:00.000+00:00
Duration: 8 min 35 s
Description: Watch this Scientific Journal Video about In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells at JoVE.com

这项工作得到了NIAMS AR064873，Epigen Project PB的支持。 P01.001.019 / Progetto Bandiera Epigenomica IFT to LL

根据标准动物设施程序培育和维持小鼠，所有实验方案均经过动物福利保证和意大利卫生部内部动物研究伦理委员会批准，并遵守"NIH护理和使用指南"实验动物。 肌肉损伤和自噬通量的体内阻滞<ol><li> 肌肉受伤。 <ol><li>为了诱导急性骨骼肌损伤，将20μL的10μM心脏毒素（CTX）直接注射到体重约为20g的2个月大的C57BL / 6J小鼠的左胫骨前部（TA）肌肉中。使用相等数量的男性和女性。使用不受干扰的对侧肢体作为对照。 注意:在0.9％氯化钠溶液中的10μMCTX应过滤（0.22μmPVDF过滤器）并储存在-20°C。避免反复冻融循环。 </li><li>使用我具有30G针头的nsulin注射器，在TA的中间注射CTX。为了在TA中引起精确的伤害，确保注射器的针进入从肌肉的底部到顶部5mm深的远端肌腱附近（ 图1A ）。 </li></ol></li><li> 在不受干扰和受伤的小鼠中阻断自噬通量。 <ol><li>为了评估自噬通量，24小时后损伤（pi）通过腹膜内注射（IP）每24小时给予50mg / kg氯喹（CLQ）4天（ 图1B ）。 <ol><li>将CLQ（10mg / mL）溶于PBS 1X中并过滤0.2μm膜。称量每只老鼠，计算注射量的CLQ。 注意:在同一天准备和使用CLQ解决方案。 CLQ库存可在-20°C下储存长达一个月。由于自噬是一种与代谢有关的过程，因此总是在同一时间执行CLQ治疗（ 即早晨befo）10 AM）。 </li></ol></li></ol></li></ol>肌肉组织切片<ol><li> 老鼠牺牲 <ol><li>损伤后5天对小鼠进行安乐死。 </li><li>通过颈椎脱位或CO 2进行安乐死（随后进行颈椎脱位确认）。由于自噬过程与小鼠睡眠/饮食习惯有关，因此始终同时牺牲小鼠，优选在早晨（ 即早上10点之前）。 </li></ol></li><li> TA隔离和包容。 <ol><li>解剖前，用70％乙醇喷雾小鼠。 </li><li>使用剪刀在臀部水平的小鼠的背部皮肤上做一个小的垂直切口（3毫米长）。切割尾巴和脚部以简化皮肤清除。将皮肤拉向尾部，并将其取出以暴露下面的肌肉; TA肌肉容易定位， 如图2A所示 </STRONG&gt;。 </li><li>在两个镊子的帮助下，抓住远端肌腱。 </li><li>切远端肌腱; TA和伸肌腱（EDL）肌腱通常被共同剪切并分开（见步骤2.2.6）。 </li><li>使用镊子，用肌腱保持TA肌肉，并小心地将肌肉拉近近端（靠近膝盖; 图2B ）。 注意:此时，EDL和TA肌肉易于识别，TA比EDL更大，更肤浅。 </li><li>通过沿相反方向拉拽两根远端肌腱将TA与EDL肌肉分开（ 图2C ）。 </li><li>切开近端肌腱。 </li><li>使用镊子，去除覆盖肌肉的薄筋膜，而不会损坏组织。 </li><li>借助纸巾去除样品中的过多水分。确保肌肉既不潮湿也不会过度干燥，因为过多的水分会产生信号对肌肉造成重大损害，危及下一次染色。 </li><li>将最佳切割温度（OCT）化合物放置在模具底部，并将肌肉放入其中，方向如图2D所示。向烧杯中加入100％异戊烷，直至达到约3-4cm的深度。将放置在聚苯乙烯箱中的烧杯与液氮接触。 </li><li>不要让液氮进入烧杯，因为它会产生一个泡沫泡沫，这可能会影响夹杂物并损伤肌肉部分。 </li><li>观察异戊烷，并等到达到适当的温度（-140°C和-150°C之间）;在适当的温度下，冷却​​的异戊烷的固体白色层将在烧杯的底部结晶。 </li><li>如果异戊烷完全冻结固体，熔化并冷却至冷冻温度，然后再进行下一步骤。 </li><li>使用预制镊子，精细地将模具降低异戊烷约20-30秒。将冷冻的样品放入带有干冰的容器中，直至转移到-80°C的冷冻箱中。 注意:协议可以在这里暂停。 </li></ol></li><li> 肌肉组织冷冻切片。 <ol><li>在-80°C至少一夜之后，进行冷冻切片。 </li><li>将低温恒温刀和防滚板从-20°C冷冻箱中取出，并将它们放在相应的支架上进入低温恒温柜。 </li><li>将OCT上的一滴OCT放在样品柱上，并将其放置在垂直NS方向的冷冻样品上， 如图2D所示 。 </li><li>将样品桩放在卡盘上。 </li><li>不要将抗卷板向下拉在刀上，在40微米处做一些切割，以摆脱不包括肌肉的OCT。当肌肉变得可见时，将切片厚度减小到7-8μm。 </li><li>拉下防卷板直到与其对齐刀的边缘或一点点以上。 </li><li>切割7-8μm厚的横切面，每个组织学载玻片安装3-4片。 注意:建议的低温恒温器温度在-15°C和-23°C之间。样本的横截面需要随后的分析来确定卫星位置的肌肉干细胞。 </li><li>为了使切片的粘附最大化，将载玻片在室温下保持10-15分钟，以防止其在抗体孵育过程中脱落。 注意:空气干燥步骤可能会影响免疫染色，提供不明确的结果。幻灯片可以在-80°C下保存几个月。协议可以在这里暂停。 </li></ol></li><li> 通过进行苏木精曙红（H＆E）染色检查TA肌肉冷冻切片中的肌肉损伤。 <ol><li>评估肌肉的质量（ 即损伤的有效性，肌肉的分离和包容离子和冷冻切片），进行H＆E染色。对于每个实验时间点，用4％PFA固定一个幻灯片10分钟。固定后，在1x PBS的几个变化中清洗载玻片。 注意:注意。 PFA是致癌物质，必须小心处理。 </li><li>为每个实验点取一张幻灯片，并将其放入染色罐中。 </li><li>用9克/升苏木精填充罐，直到切片被覆盖并孵育8分钟。 </li><li>回收苏木精。 </li><li>将瓶子放在流水下10分钟，以除去过量的苏木精。 注意:确保不要将水直接流入各部分。 </li><li>用无菌水冲洗。 </li><li>在酸化的90％乙醇中孵育0.5％（w / v）曙红1分钟。 </li><li>回收曙红。 </li><li>用无菌水洗涤两次，洗涤3分钟。 注意:在化学防护罩上执行以下步骤。 </li><li>用70％乙醇洗涤切片。 </li><li>洗具有90％乙醇的切片。 </li><li>用100％乙醇洗涤切片。 </li><li>使用0.879 g / mL邻二甲苯孵育切片。 注意:注意。邻二甲苯是易燃和中等毒性的。在化学防护罩下操作，戴防护装备，包括手套，实验室外套和面罩。 </li><li>回收o-二甲苯。 </li><li>将幻灯片放在一张纸上干燥。 </li><li>使用快速硬化的基于二甲苯的安装介质封闭载玻片。 </li><li>以10倍放大倍率在光学显微镜下检查切片的质量（ 见图3 ） 注意:协议可以在这里暂停。 </li></ol></li></ol> 3.在损伤的肌肉组织切片中LC3和MyoD的免疫染色<ol><li> 肌肉组织切片的固定。 <ol><li>通过润湿一张纸并将其放置在可关闭的塑料盒的底部以保持较高的温度来准备孵育室室内湿度很大。将组织切片用1％PBS中的4％多聚甲醛（PFA）固定10分钟。 注意:使用湿纸张以避免样品干燥。准备新鲜的PFA或使用储存在-20°C的PFA。警告。 PFA有毒;制造原料溶液时，必须小心处理粉末。该操作必须在化学防护罩上进行，并带有防护装备，包括手套，实验室外套和面罩。此外，在解决方案时，应仔细操作PFA。 </li><li>取出PFA，用1x PBS洗涤5〜7分钟;重复这一步3次。 </li></ol></li><li> 肌肉组织切片的渗透。 <ol><li>使用pap笔在组织切片周围绘制疏水屏障。 注意:此步骤定义了组织切片周围的表面，并使步骤3.4,3.6,3.7和3.9中描述的抗体体积最小化。 </li><li>用200μL冷（-20°C）覆盖切片，甲醇，并将孵育室水平置于-20℃的冷冻箱中5分钟。 </li><li>从冷冻箱中取出孵育室，抽出甲醇，并在室温下用1X PBS洗涤切片5-7分钟;重复此步骤3x。 </li></ol></li><li> 闭塞 <ol><li>准备4％牛血清白蛋白（BSA）在PBS 1X中的新鲜块溶液。 注意:BSA溶液可以在4℃下储存1周。 </li><li>在室温下用块溶液孵育至少60分钟。 </li><li>移除阻塞解决方案。 </li><li>通过稀释1×PBS中的20μg/ mL抗Fab制备100μL抗Fab混合物。使用非偶联亲和纯化的抗小鼠IgG的F（ab）片段在室温孵育切片1小时。 </li></ol></li><li> 初级抗体孵育。 <ol><li>通过将LC3和MyoD抗体溶解在1％PBS中的4％BSA中，为每个载玻片制备100μL的一抗混合物。使用5μg/mL抗LC3（兔多克隆抗体）和10mg / L抗MyoD（小鼠单克隆抗体）。在4℃孵育一次抗体混合物过夜。 </li></ol></li><li> 洗。 <ol><li>取出一级抗体混合物。 </li><li>在1X PBS中用1％BSA洗涤切片10分钟;重复此步骤3x。 </li></ol></li><li> 二次抗体孵育。 <ol><li>为每个载玻片准备100μL的二抗混合物。在1％PBS中的4％BSA中溶解山羊抗兔488（5μg/ mL）和山羊抗小鼠594（5μg/ mL）。 注意:避免过度的光照，以防止荧光染色。在黑暗中执行以下步骤。 </li><li>在室温下使用二级抗体混合物孵育切片45分钟。 </li><li>取出第二抗体混合物，用1x PBS洗涤切片5-7分钟;重复此步骤3x。 </li></ol></li><li> 初级抗体孵育。 <ol><li>稀释层压板在1％PBS中的4％BSA中的n-2（α-2-链）单克隆抗体（0.33μg/ mL），每个载玻片使用100μL混合物。在一次抗体混合物中孵育1-2小时。 </li></ol></li><li> 洗。 <ol><li>取出一级抗体混合物。 </li><li>在1X PBS中用1％BSA洗涤切片10分钟;重复此步骤3x。 </li></ol></li><li> 二次抗体孵育。 <ol><li>通过在1％PBS中的4％BSA中稀释山羊抗大鼠647（5μg/ mL），为每个载玻片制备100μL的二抗混合物。在二级抗体混合物中孵育45分钟。 </li><li>取出第二抗体混合物，用1x PBS洗涤切片5-7分钟;重复此步骤3x。 </li></ol></li><li> 4&#39;，6-二脒基-2-苯基吲哚（DAPI）孵育。 <ol><li>在每个载玻片中加入200μL300 nM DAPI溶液，在1×PBS中。在室温下孵育5分钟。将DAPI溶液储存在4℃黑暗。 </li><li>用1x PBS洗涤切片5-7分钟;重复此步骤3x。 </li></ol></li><li> 安装染色肌肉部分。 <ol><li>从部分去除多余的洗涤溶液。 </li><li>将10μL甘油在1×PBS（3:1）中放置在载玻片中间的染色部分，并加入盖玻片，避免形成气泡。 </li></ol></li></ol>共聚焦显微镜获取<ol><li>使用与图像采集系统和分析软件集成的四激光共焦显微镜获取图像。 注意:MyoD与594染料（一种亮红色荧光染料）共轭，其激发理想地适用于594nm激光（激发:590nm，发射:617nm）。 LC3与488染料（其是亮绿色荧光染料）共轭，理想地适用于488nm激光线（激发:495nm，发射:519nm）。层粘连蛋白与647染料缀合一种明亮的远红荧光染料，具有理想适用于647nm激光线（激发:650nm，发射:668nm）的激发。 </li><li>将幻灯片放置在显微镜托盘中并使用放大倍数63倍以检测核信号。通过使活动再生的领域居中，依靠肌肉形态，手动聚焦在再生区域（参见图4 ）。 注意:在无刺激的肌肉中，肌纤维的大小几乎是恒定的（ 图4A ），损伤介导的肌肉再生可以通过更新的肌纤维的外观来识别，这是再生后新形成的纤维。此外，主动再生中的肌肉的特征在于由巨噬细胞，纤维成脂前体以及定位于受损肌肉以编排肌肉再生的细胞形成的广泛浸润（ 图4B ）。 </li><li>评估再生中的免疫荧光染色重点是位于肌纤维基底层下的MuSCs。 </li><li>专注于对MyoD染色阳性的MuSCs，并位于由层粘连蛋白染色标记的肌纤维内（参见图4B ，白色箭头）。 </li><li>使用至少3只小鼠/实验组，并使用显微镜获得每个实验组至少7个场的63倍放大图像。 </li><li> 一旦识别再生区域，使用DAPI染色进行聚焦，然后调整其他单个通道。 <ol><li>使用以下显微镜设置:通道A594针孔1.2通风单元，增益791;通道A488针孔1.6通风单位，增益659;通道A647针孔1.4通风单位，增益719。 </li></ol></li><li>调整单个通道的焦点后，继续采集1,024 x 1,024帧大小和12.61μs像素停留的图像。 </li><li>计算MyoD阳性细胞的百分比（对照t他正确地位于层下）LC3阴性和显示LC3信号的MyoD阳性细胞的百分比。 注意:显示LC3染色的MyoD阳性细胞的百分比是该方案的读数。 </li></ol>

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作者没有什么可以披露的

<html>该协议描述了如何在代偿性肌肉再生期间监测骨骼肌干细胞中的自噬。尝试了几种用于LC3和MyoD共染色的抗体，在这里列出了在小鼠组织切片中工作并产生成功结果的抗体 （参见材料表 ）。强烈推荐使用甲醇渗透（见步骤3.2.2）以进行成功染色。 该方案的局限性与小鼠的内在变异性相关，强迫使用至少3只小鼠/实验组。该方法反映了在骨骼肌再生...

骨骼肌再生是成体干细胞（肌肉卫星细胞，MuSCs）与参与再生过程的其他细胞类型之间相互作用的结果。肌肉的体内平衡和功能由肌肉利基和全身线索1,2所组合的信号保持。在整个一生中，已经报告了MuSC功能，肌肉利基和系统线索的变化，导致老年人的功能能力下降3 。 MuSCs位于基底层下方的一个利基，并且在肌肉损伤时被激活以修复损伤的肌肉4,5 。为了确保有效的再生反应，至关重要的是，MuSC协调退出静止，自我更新和增殖扩张阶段所需的不同过程通过肌原性分化6 。在老年人和肌肉慢性疾病中，所有这些功能受到损害，导致肌肉功能变化2,3,6,7,8,9,10,11,12,13。 Macroautophagy（以下称为自噬）正在成为维持组织稳态至关重要的生物过程14 。自噬过程包括贩运机制，其中细胞质，细胞器和蛋白质的部分被吞入囊泡，其最终通过溶酶体途径降解，促进有毒分子的去除和大分子的再循环cules。这提供了能量丰富的化合物，以支持在应激或其他不利条件下的细胞和组织适应15,16 。与其细胞存活活动一起，自噬也可以作为细胞死亡诱导剂，取决于细胞组织背景（ 例如，正常与癌组织）和应激刺激的类型17,18 。 最近的证据表明，自噬需要维持肌肉质量和肌纤维完整性19,20 ，据报道在不同的肌营养不良21,22,23 中受损，包括Duchenne肌营养不良症（DMD）24,25,26,27 </su同样，在失去肌肉量（称为肌肉减少症）后，31,33,34,35岁的老年人中观察到自噬过程的逐渐减少32,33,34 ， 35,36,37和肌纤维存活38 。 热带实验室的一项研究预计自噬和骨骼肌再生潜能之间有着密切的关系，这表明热量限制增强了MuSC的可用性和活性。这不行最近观察到Foxo3-Notch轴激活了自我更新期间的自噬过程40以及从静止到增殖状态的MuSC转变。这些数据符合青年时期老年人和老年人的基础性自噬的逐渐减少，以及老年期间MuSCs的数值和功能下降42 。 在最近的一篇论文中，我们展示了自噬和补偿性肌肉再生之间的密切关系，区分了DMD进展的早期阶段。因此，当肌肉再生受损和纤维化组织沉积发生时，我们观察到疾病进展后期阶段的自噬通量减少。有趣的是，我们表明，在再生条件下，自噬在MuSCs中被激活，并且调节自噬过程影响MuSC激活和fu功能30 总而言之，这些数据突显了在正常和病理状况下以及整个寿命期间，在肌肉再生过程中探索MuSCs自噬过程的紧迫性。在这里，我们提供了一个协议，通过对微管相关蛋白1A / 1B-轻链3（LC3） 进行原位免疫染色监测肌肉再生条件下的自噬过程，LC3是自噬标志物43 ，MyoD是一种标记肌原纤维谱系，在对照组和受损小鼠的肌肉组织切片中。报告的方法允许监测一个特定细胞室的自噬过程，MuSC在编排肌肉再生中起关键作用。

<table><tbody><tr><td>C57BL/6J</td><td>The Jackson Laboratory</td><td>000664</td><td>WT mice</td></tr><tr><td>Cardiotoxin 1</td><td>Latoxan</td><td>L8102</td><td></td></tr><tr><td>Millex-VV</td><td>Merck Millipore</td><td>SLVV033RS</td><td>Syringe Filter Unit, 0.1 &amp;micro;m, PVDF, 33 mm, gamma sterilized</td></tr><tr><td>Chloroquine diphosphate salt</td><td>Sigma-Aldrich</td><td>C6628</td><td>Caution:&lt;br/&gt; Harmful if swallowed</td></tr><tr><td>BD Micro-Fine + 0.5 mL</td><td>BD</td><td>324825</td><td></td></tr><tr><td>Tissue-Tek O.C.T. compound</td><td>Sakura Finetek</td><td>25608-930</td><td></td></tr><tr><td>Tissue-Tek Cryomold Intermediate</td><td>Sakura Finetek</td><td>4566</td><td></td></tr><tr><td>2-Methylbutane</td><td>Sigma-Aldrich</td><td>277258</td><td></td></tr><tr><td>Hematoxylin Solution, Harris Modified</td><td>Sigma-Aldrich</td><td>HHS32</td><td></td></tr><tr><td>Eosin Y solution, alcoholic</td><td>Sigma-Aldrich</td><td>HT110132</td><td></td></tr><tr><td>o-Xylene</td><td>Sigma-Aldrich</td><td>X1040</td><td>Caution:&lt;br/&gt; Flammable liquid and vapour; May be fatal if swallowed and enters airways; Harmful in contact with skin; May cause respiratory irritation; Causes serious eye irritation</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich</td><td>P6148</td><td>Caution:&lt;br/&gt; Flammable solid; Harmful if swallowed; Causes skin irritation; May cause an allergic skin reaction; Causes serious eye damage; May cause respiratory irritation; Suspected of causing cancer</td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Thermo Fisher Scientific</td><td>14190-094</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A7030</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Eukitt - Quick-hardening mounting medium</td><td>Sigma-Aldrich</td><td>3989</td><td></td></tr><tr><td>AffiniPure Fab Fragment Goat Anti-Mouse IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>115-007-003</td><td></td></tr><tr><td>LC3B Antibody</td><td>Cell signaling Technology</td><td>2775</td><td></td></tr><tr><td>Monoclonal mouse anti-MyoD&lt;br/&gt; (concentrated) clone 5.8A</td><td>DAKO - Agilent Pathology Solutions</td><td>M3512</td><td></td></tr><tr><td>Laminin-2 (&amp;alpha;-2-chain) monoclonal antibody</td><td>Enzo Life Sciences</td><td>4H8-2</td><td></td></tr><tr><td>Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L)</td><td>Life technologies</td><td>A11008</td><td></td></tr><tr><td>Alexa Fluor 594 Goat Anti-Mouse IgG (H+L)</td><td>Life technologies</td><td>A11005</td><td></td></tr><tr><td>Alexa Fluor Goat Anti-Rat IgM Antibody</td><td>Life technologies</td><td>A21248</td><td></td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Thermo Fisher Scientific</td><td>D1306</td><td></td></tr></tbody></table>

in situ immunofluorescent staining of autophagy in muscle stem cells

越来越多的证据表明自噬是维持组织稳态的重要调节过程。已知自噬涉及骨骼肌发育和再生，并且自噬过程已经在几种肌肉病理学和年龄相关的肌肉疾病中描述。与肌肉修复期间卫星细胞的功能衰竭相关的自噬过程的最近描述的块支持活性自噬与生产性肌肉再生相结合的概念。这些数据揭示了在正常和病理状态（如肌营养不良）的肌肉再生过程中自噬在卫星细胞活化中的关键作用。在这里，我们提供了在肌肉再生条件下监测成年肌肉干细胞（MuSC）隔室中的自噬过程的方案。该协议描述了LC3的原位免疫荧光成像的设置方法，autophagy标记和MyoD，一种肌原性谱系标记，来自对照组和受损小鼠的肌肉组织切片。报告的方法允许监测一个特定细胞室的自噬过程，MuSC隔室在调节肌肉再生中起着核心作用。

该方案描述了在肌肉再生过程中检测MuSCs中的自噬的有效的原位方法。 CTX 体内治疗: 使用CTX诱导TA肌肉的肌肉损伤，并使用不受干扰的肌肉作为对照。由于自噬是高度动态的，通过执行CLQ的IP注射来阻断自噬通量（ 图1 ）。 CLQ治疗对于评估自噬通?...

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In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

活动性自噬与生产性肌肉再生有关，肌肉再生对肌肉干细胞（MuSC）的激活至关重要。在这里，我们提供了原位检测LC3的方案，这是来自对照组和受伤小鼠的肌肉组织切片的MyoD阳性MuSC中的自噬标记。

肌肉干细胞中自噬的原位免疫荧光染色

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

in-situ-immunofluorescent-staining-of-autophagy-in-muscle-stem-cells

Research

JoVE Journal

Biology

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

10.1K 次观看.  Italian National Research Council. 活动性自噬与生产性肌肉再生有关，肌肉再生对肌肉干细胞（MuSC）的激活至关重要。在这里，我们提供了原位检测LC3的方案，这是来自对照组和受伤小鼠的肌肉组织切片的MyoD阳性MuSC中的自噬标记。 

视频: In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

科研

发育生物学

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Pax7 和层粘连蛋白抗体免疫荧光法鉴定骨骼肌卫星细胞

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

采用先进的3D荧光显微镜定量分析自噬

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

生物学

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 ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

活细胞成像早期自噬活动：Omegasomes与超越

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

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 ...

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome and are recycled. During autophagy, cytoplasmic LC3 protein is lipidated and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to form the autolysosome, where the breakdown of the autophagosome vesicle and its contents occurs. The ubiquitin-associated protein p62, which binds to LC3, is also used to monitor autophagic flux. Cells undergoing autophagy should demonstrate the co-localization of p62, LC3, and lysosomal markers. Immunofluorescence microscopy has been used to visually identify LC3 puncta, p62, and/or lysosomes on a per-cell basis. However, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, multispectral imaging flow cytometry was used along with an analytical feature that compares the bright detail images from three autophagy markers (LC3, p62 and lysosomal LAMP1) and quantifies their co-localization, in combination with LC3 spot counting to measure autophagy in an objective, quantitative, and statistically robust manner.

Here, multispectral imaging flow cytometry with an analytical feature that compares bright detail images of 3 autophagy markers and quantifies their co-localization, along with LC3 spot counting, was used to measure autophagy in an objective, quantitative, and statistically robust manner.

通过使用多光谱成像流式细胞术测量LC3，p62和LAMP1共定位评估自噬通量

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via ...

Immunolabelling Myofiber Degeneration in Muscle Biopsies

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. Therapeutic options addressing the causes of muscular dystrophy pathogenesis are expected to alleviate muscle degeneration. Therefore, a method to assay and quantify the extent of cell death in muscle biopsies is needed. Conventional methods to observe myofiber degeneration in situ are either poorly quantitative or rely on the injection of vital dyes. In this article, an immunofluorescence protocol is described that stains necrotic myofibers by targeting immunoglobulin G (IgG) uptake by myofibers. The IgG uptake method is based on cell features characterizing the necrotic demise, including 1) the loss of plasma membrane integrity with the release of damage-associated molecular patterns and 2) the uptake of plasmatic proteins. In murine cross-sections, the co-immunolabelling of myofibers, extracellular matrix proteins, and mouse IgG allows clean and straightforward identification of myofibers with necrotic fate. This simple method is suitable for quantitative analysis and applicable to all species, including human samples, and does not require the injection of vital dye. The staining of necrotic myofibers by IgG uptake can also be paired with other co-immunolabelling.

Described here is a protocol for direct immunolabelling of necrotic myofibers in muscle cryosections. Necrotic cells are permeable to serum proteins, including immunoglobulin G (IgG). Revealing the uptake of IgG by myofibers allows the identification and quantification of myofibers that undergo necrosis regardless of muscle condition.

肌肉活检中的免疫标记肌纤维退化

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

单一肌纤维培养测定法用于评估体外成人肌肉干细胞功能

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

采集小鼠骨骼肌以培养干细胞及其生态位

Unfractionated Bulk Culture of Mouse Skeletal Muscle to Recapitulate Niche and Stem Cell Quiescence

Skeletal muscle is the largest tissue of the body and performs multiple functions, from locomotion to body temperature control. Its functionality and recovery from injuries depend on a multitude of cell types and on molecular signals between the core muscle cells (myofibers, muscle stem cells) and their niche. Most experimental settings do not preserve this complex physiological microenvironment, and neither do they allow the ex vivo study of muscle stem cells in quiescence, a cell state that is crucial for them. Here, a protocol is outlined for the ex vivo culture of muscle stem cells with cellular components of their niche. Through the mechanical and enzymatic breakdown of muscles, a mixture of cell types is obtained, which is put in 2D culture. Immunostaining shows that within 1 week, multiple niche cells are present in culture alongside myofibers and, importantly, Pax7-positive cells that display the characteristics of quiescent muscle stem cells. These unique properties make this protocol a powerful tool for cell amplification and the generation of quiescent-like stem cells that can be used to address fundamental and translational questions.

作者聚焦:使用生态位组分培养静止肌肉干细胞的离体方案 

Skeletal muscle comprises multiple cell types, including resident stem cells, each with a special contribution to muscle homeostasis and regeneration. Here, the 2D culture of muscle stem cells and the muscle cell niche in an ex vivo setting that preserves many of the physiological, in vivo, and environmental characteristics are described.

小鼠骨骼肌的普通散装培养物，以概括生态位和干细胞静止

Skeletal muscle is the largest tissue of the body and performs multiple functions, from locomotion to body temperature control. Its functionality and ...