这项工作得到了强直性营养不良基金会（MDF-FF-2014-0013）和美国心脏协会（16SDG29320001）的科学家发展拨款的基金A-Fellow资助部分支持。

野生型和Nestin-GFP转基因小鼠被安置在明尼苏达大学的动物设施中。所有实验程序均由明尼苏达大学体制动物护理和使用委员会批准，并符合NIH"实验动物护理和使用指南"。 肌肉解剖和单细胞隔离<ol><li>用三溴乙醇（250mg / kg，ip）将成年小鼠（6-10周，男性和女性）安乐死，并用70％乙醇对其腹部皮肤进行消毒。 注意:这里使用三溴乙醇代替氯胺酮进行麻醉/安乐死，因为已知氯胺酮与NMDA受体相互作用，这可能对研究有影响。 </li><li>将小鼠放置在仰卧位，使用手术刀在腹部皮肤上进行水平切口。用手剥离皮肤，拉动相反方向以暴露后肢肌肉。 </li><li> COLLEC使用镊子和剪刀从两个后肢的肌肉。将它们储存在补充有1％青霉素 - 链霉素（P / S）的无菌磷酸盐缓冲盐水（PBS）中。 </li><li>在补充有1％P / S的冰冷PBS中洗涤解剖后肢肌肉两次，并将其转移到无菌10cm板上。 </li><li>使用镊子和剪刀，在2倍放大倍数的夹层显微镜下，从肌肉中仔细剖析神经，血管和结缔组织。 </li><li>使用无菌剪刀和刀片将肌肉切碎并切碎成小块（1-2毫米3 ）。如有必要，加入少量Dulbecco&#39;s Modified Eagle Medium（DMEM），以确保肌肉不会变干。 </li><li>通过10毫升血清移液管上下移液10次机械分解组织。 </li><li>加入新鲜消化溶液（补充有0.2％2型胶原酶的DMEM）。在37℃下用香茅孵育2小时以35转/分钟搅拌。 </li><li>使用18 G针头研磨以均匀化混合物。然后，以500×g离心5分钟。弃去上清液，然后将沉淀重悬于0.25％胰蛋白酶/ EDTA。在37℃孵育10分钟。重复离心步骤两次。 </li><li>加入10 mL补充有20％胎牛血清（FBS）的DMEM溶液，并以500 xg离心5 min。弃去上清并将沉淀重悬于红细胞裂解缓冲液（155mM NH 4 Cl，10mM KHCO 3和0.1mM EDTA）中。 </li><li>以500 xg离心5分钟。弃去上清液并将沉淀物重新悬浮在分选缓冲液（20mM HEPES，pH 7.0; 1mM EDTA;和1％BSA，不含Ca / Mg 2+的 PBS，pH 7.0）中。通过40μm的细胞过滤器过滤混合物，以获得单细胞悬液。 </li><li>以500 xg离心5分钟。弃去上清液，并将沉淀重悬于1 mL分选缓冲液中。 </li><li>算了细胞数用血细胞计数器，并在分选缓冲液中将单细胞悬浮液稀释至5×10 6 / mL。 </li></ol>细胞染色和分选<ol><li>按照表1所述准备对照和样品。如表1所述，用冰上各自的抗体将单细胞悬浮液染色30分钟。 </li><li>以500×g离心5分钟，用分选缓冲液洗涤两次。 </li><li>将DAPI添加到单细胞溶液中，如表1所示。对于PDGFRβ-PE-FMO对照和样品，使用5μg/ mL DAPI（终浓度）作为DAPI单色对照和1μg/ mL DAPI。在整个实验过程中将所有管保持在冰上。 </li><li>打开分拣机和软件。提示时，扫描并插入100μm的分选芯片。 </li><li>执行自动设置（ 即，芯片对齐，液滴校准，侧流校准和排序延迟校准on）通过在提示时加载自动设置珠。 </li><li>自动设置完成后，进入"实验"选项卡，点击"新建"，从"公共模板"中选择"空白模板"。 </li><li>在"测量设置"下，输入"FL1"，"FL2"的"Nestin-GFP"和"FL3"的"PDGFRβ"的"DAPI"。取消选中"FL4" - "FL6"的框。 </li><li>勾选方框激活405,448和561激光，然后点击"创建新实验"。 </li><li> 选择"开始补偿向导"选项，并按照"补偿向导"软件提示设置补偿。 <ol><li>加载未染色的控件，然后单击"开始"。点击"检测器和阈值设置"，并调整FSC和BSC检测器的传感器增益，使人口达到规模。 </li><li>广告只是FL1-FL3荧光通道的增益水平将负个体放在直方图的左侧。点击"记录"按钮记录数据。 </li><li>出现提示时逐个加载单色控件。单击"开始和记录"以记录数据。调整直方图上阳性人群的门。点击下一步。" </li><li>转到"补偿"选项卡上的"计算矩阵"，然后单击"计算补偿设置"面板中的"计算"，执行补偿。单击"完成"退出"补偿向导"。 </li></ol></li><li>加载PDGFRβ-PE-FMO控件，然后单击"开始"。在"所有事件"图下的感兴趣单元周围绘制多边形门（门A）。 </li><li>双击门A内创建一个小孩绘图。将Y轴更改为DAPI并在活（DAPI 低 ）单元周围绘制多边形门（Gate B）。双击我N侧门B创建一个小孩情节。将X轴更改为FSC-H，将Y轴更改为FSC-W，并在单体周围绘制多边形门（Gate C）以消除双峰。 </li><li>双击门C内创建一个小孩图。将X轴更改为Nestin-GFP，将Y轴更改为PDGFRβ-PE。点击"记录"按钮记录数据。 </li><li>加载样品并重复步骤2.11-2.12。录制后，点击"暂停"保存样品。 </li><li>基于PDGFRβ-PE-FMO对照定义PDGFRβ +和Nestin-GFP +细胞的门控边界。绘制PDGFRβ + Nestin-GFP 和 PDGFRβ + Nestin-GFP +群体的门。 </li><li>在"排序方法"下，选择"双向管"，将"PDGFRβ + Nestin-GFP"和"PDGFRβ + Nestin-GFP + "细胞分配到左右收集管，spectively。将分选缓冲液填充的15 mL收集管装入收集台上，然后单击"加载收集"按钮。 </li><li>单击"恢复"按钮保持样品运行。单击"开始排序"以收集PDGFRβ + Nestin-GFP - （I型周细胞）和PDGFRβ + Nestin-GFP + （II型周细胞）细胞。 </li></ol>后排序分析<ol><li>将分选的细胞以500×g离心5分钟，将沉淀重悬于1mL周细胞培养基中（参见材料表 ），并使用血细胞计数器计数细胞密度。 </li><li>在约1×10 4个细胞/ cm 2的聚-D-赖氨酸（PDL）涂覆的盖玻片上的种子I型和II型周细胞。在37℃下用5％CO 2在周细胞培养基中生长3天。 </li><li>在第3天，使用荧光微观检查周细胞形态（相位对比）和内源性巢蛋白-GFP表达范围（激发激光:488nm，激发滤光片:470/40nm，发射滤光片:515 / 30nm）。以20X物镜（0.45 NA）拍摄图像。 </li><li>如前所述，分别用脂肪生成（小鼠MSC基础培养基+脂肪形成刺激性补充剂）和肌原性（DMEM + 2％马血清）培养基替代周细胞培养基，以启动脂肪形成和肌原性分化。每2-3天更换培养基。 </li><li>在室温下，在4％多聚甲醛（PFA）中，在17天（脂肪形成/肌原成分分化后14天）将细胞固定20分钟。 注意:注意，PFA是致癌物质。 </li><li> 如先前的出版物19,20所述，对Perilipin（脂肪细胞标记）和S-肌球蛋白（成熟肌管/肌纤维标记）进行免疫细胞化学。 <ol><li>在PBS中将固定细胞洗涤3次，室温10分钟。 </li><li>加入封闭缓冲液（PBS补充5％驴血清，3％BSA和0.3％Triton X-100），并在室温下孵育1小时。 </li><li>在4℃下将细胞与抗Perilipin（2μg/ mL）和/或抗S-肌球蛋白（2μg/ mL）抗体孵育过夜。 </li><li>在PBS中将细胞洗涤3次，室温10分钟。 </li><li>在室温下使用Alexa 555驴抗兔（4μg/ mL）和/或Alexa555驴抗小鼠（4μg/ mL）抗体孵育细胞1小时。 </li><li>在PBS中将细胞洗涤3次，室温10分钟。 </li><li>用含有DAPI的安装介质装载免疫染色细胞（参见材料表）。使用荧光显微镜（激发激光:543nm; 540 / 45nm激发和600/50nm发射）检查肾上腺素和S-肌球蛋白表达，并在40X物镜（0.60NA）下拍摄图像。 </li></ol></li></ol>

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所有作者没有利益冲突的披露。

周细胞是位于毛细血管21,26的外侧表面上的多能血管周围细胞22,23 。在骨骼肌中，周细胞能够沿着脂肪生成和/或造血途径19,20,24区分。最近的研究揭示了两个亚细胞周期细胞，具有不同的标记表达和不同的分化潜力19,24,25。 I型（NG2 + Nestin - ）周细胞是脂肪形成的，而II型（NG2 + Nestin + ）周细胞是肌原性的。这些亚群体外研究的分离需要NG2-DsRed和Nestin-GFP双转基因小鼠品系。这种净化的依赖性双转基因小鼠的方案显着限制了其应用，从而对周细胞亚群的生物学进行了研究。 该视频文章说明了从小鼠骨骼肌分离I型和II型周细胞的另一种方法。这种新方法仍然依赖于基于FACS的细胞分离技术。然而，代替利用转基因NG2-DsRed荧光，其靶向内源性PDGFRβ信号。这是基于观察到NG2-DsRed表达与骨骼肌中的PDGFRβ共定位19 。与原始方法相比，该协议具有几个优点。首先，它不需要NG2-DsRed遗传背景，尽管仍然需要Nestin-GFP背景。第二，该方案不是针对NG2，而是使用经证实和可商购的抗体的良好表征的标记物PDGFRβ。第三，它允许同时隔离博th型I型和II型周细胞。例如，使用该方案分离的PDGFRβ-PE + Nestin-GFP-和PDGFRβ-PE + Nestin-GFP +细胞表现出明显的分化能力。具体来说，PDGFRβ-PE + Nestin-GFP，而不是PDGFRβ-PE + Nestin-GFP + ，在成脂条件下分化成脂肪细胞，而PDGFRβ-PE + Nestin-GFP + ，而不是PDGFRβ-PE + Nestin-GFP- ，细胞在肌原性条件下经历肌原性分化。这些数据与以前的报道一致，显示NG2-DsRed + Nestin-GFP-细胞（I型周细胞）是脂肪形成的，NG2-DsRed + Nestin-GFP +细胞（II型周细胞）是肌原性19,24，表明分离的PDGFRβ -PE + Nestin-GFP和PDGFRβ-PE + Nestin-GFP +细胞分别确实是I型和II型周细胞。一起，这些结果表明，这个新的方案允许从鼠骨骼肌和可能的其他组织相对容易地分离/纯化周细胞亚群。这将促进关于周围细胞生物学及其各种疾病的治疗性翻译的研究。 然而，应注意，由于使用PDGFRβ，该方法存在限制。在先前的研究中已经显示PW1 +间质细胞（PIC）也表达PDGFRβ20。因此，分离的PDGFRβ-PE + Nestin-GFP和PDGFRβ-PE + Nestin-GFP +群体可能含有PIC。然而，这些PIC对周边细胞分化的贡献是有限的，因为周边细胞超过骨骼肌20中的 PIC，而I型周细胞不经历发生19,25。 使用该方法，分别获得了分别为9.5％和2.1％的I型和II型周细胞。这些数字与双转基因小鼠中分别报道的产量分别为2.8％和3.4％相当。轻微的差异可能是由于不同的门控策略或消化方案。这些结果再次表明，所提出的方案可用于从骨骼肌分离周细胞的亚型。 该方案的关键步骤包括以下几点:（1）确保骨骼肌完全切碎，并能容易地通过10 mL血清移液管。大的肌肉块干扰酶消化并显着降低产量。 （2）使用新鲜的胶原酶溶液进行肌肉消化，并在孵育期间轻轻搅动（35转/分钟）。 （3）保持控制和样品在冰上整个染色和分选步骤。 （4）使用FMO控制设置门控边界。 除了分化能力外，I型和II型周细胞也具有不同的形态。具体来说，I型周细胞具有圆形细胞体，具有短的过程和相对较大的核。另一方面，II型周细胞通常具有小的细胞体，具有长而薄的过程和小的细胞核。形态差异表明，I型和II型周细胞本质上不同，这与外周细胞21的异质性一致。导致/维持I型和II型周细胞之间的差异以及潜在的分子机制仍不清楚，需要进一步调查。这个隔离协议将有助于回答这些重要问题。

肌营养不良是一种肌肉退行性疾病，目前尚无有效治疗。促进组织再生的疗法的发展一直是非常有意义的。损伤后组织再生和修复取决于常驻干细胞/祖细胞1 。卫星细胞承诺造血前体细胞有助于肌肉再生2,3,4,5,6,7。然而，他们的临床使用受到其有限的迁移和注射后的低存活率以及在体外扩增后其分化能力降低8,9,10,11的阻碍。除了satellite细胞，骨骼肌也含有许多其他具有肌原性潜能12,13,14,15,16等细胞群，如血小板衍生生长因子受体-β（PDGFRβ） - 阳性间质细胞。有证据表明，肌肉衍生的PDGFRβ +细胞能够分化成肌原细胞，改善肌肉病理和功能14,17,18,19,20。 PDGFRβ主要标记周细胞21 ，它们是多能性的血管周围细胞22,23 。除PDGFRβ之外，许多其他标志物，包括神经元胶质细胞2（NG2）和CD146也被用于齿状细胞21 。然而，应该注意的是，这些标记物都不是percyte特异性21 。最近的研究揭示了肌肉周细胞的两种亚型，称为I型和II型，其表达不同的分子标记并实现不同的功能19,24,25。生物化学上，I型周细胞是NG2 + Nestin，而II型周细胞是NG2 + Nestin + 19,24。功能上，I型周细胞可能经历脂肪分化，有助于脂肪堆积和/或纤维化，而II型周细胞可以沿着肌原性途径分化，从而有助于肌肉再生19,24,25。这些结果表明:（1）I型周细胞可能be针对脂肪退化性疾病/纤维化的治疗，（2）II型周细胞对肌营养不良具有很大的治疗潜力。这些人群的进一步调查和表征需要能够以高纯度分离I型和II型周细胞的分离方案。 目前，周细胞亚群的分离取决于NG2-DsRed和Nestin-GFP双转基因小鼠19,24。 NG2-DsRed小鼠的可用性和大多数NG2抗体的质量限制了该方法的广泛使用。鉴于所有NG2 +周细胞也在骨骼肌19,20,24中表达PDGFRβ，我们假设NG2可被PDGFRβ替代以分离周细胞及其亚群。这项工作描述了基于FACS的协议使用PDGFRβ染色和Nestin-GFP信号。这种方法对于研究者来说要求较低，因为:（1）它不需要NG2-DsRed背景，（2）它使用市售的PDGFRβ抗体，其被良好表征。此外，它能够以高纯度同时分离I型和II型周细胞，从而进一步研究这些周细胞亚群体的生物学和治疗潜力。纯化后，这些细胞可以在培养物中生长，并且它们的形态可以被可视化。这项工作还表明，使用这种方法分离的I型和II型周细胞，如从双转基因小鼠纯化的那些，分别是脂肪形成和肌原性的。

<table border="0" cellpadding="0" cellspacing="0" width="705">
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			<td height="22" style="height:22px;width:311px;">Mounting Medium with DAPI</td>
			<td style="width:173px;">Vector Laboratories</td>
			<td style="width:133px;">H-1200</td>
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</table>

isolation of type i and type ii pericytes from mouse skeletal muscles

周细胞是血管内多能细胞，其表现出不同器官甚至在相同组织内的异质性。在骨骼肌中，至少有两个周细胞亚群（称为I型和II型），其表达不同的分子标记并具有明显的分化能力。使用NG2-DsRed和Nestin-GFP双转基因小鼠，成功分离了I型（NG2-DsRed + Nestin-GFP）和II型（NG2-DsRed + Nestin-GFP + ）周细胞。然而，这些双转基因小鼠的可用性阻止了该纯化方法的广泛使用。这项工作描述了一种替代方案，允许从骨骼肌容易和同时隔离I型和II型周细胞。该协议采用荧光激活细胞分选（FACS）技术，并将靶蛋白GFP信号靶向PDGFRβ而不是NG2。隔离后，键入I和type II周细胞显示不同的形态。此外，用这种新方法分离的I型和II型周细胞，如分离自双转基因小鼠的那些，分别是脂肪形成和肌原性的。这些结果表明，该方案可用于从骨骼肌和可能来自其他组织中分离周细胞亚群。

FACS参数，包括激光强度和通道补偿，基于未染色控制和单色控制的结果进行校正。 PDGFRβ-PE-FMO对照用于设置PDGFRβ-PE +群体的门控（ 图1A ）。在PDGFRβ-PE-细胞中，代表巢蛋白-GFP +和巢蛋白 - GFP-细胞的两个群体被清楚地分离（ 图1A ）。基于PDGFRβ-PE +和巢蛋白-GFP +的门控设置PDGFRβ-PE +巢蛋白-GFP +和PDGFRβ-PE +巢蛋白-GFP -群体的门控边界（ 图1A ）。这些边界用于定义和分类PDGFRβ-PE +巢蛋白-GFP + （II型周细胞）和PDGFRβ-PE +巢蛋白-GFP-（I型周期蛋白es）细胞（ 图1B ）。分离的PDGFRβ-PE + Nestin-GFP和PDGFRβ-PE + Nestin-GFP +细胞分别占单细胞溶液中总细胞的9.5％和2.1％。 FACS分离的I型和II型周细胞在培养3天后显示出形态学差异。类型I周细胞显示变形虫形态，具有圆形细胞体和短过程（ 图2 ）。然而，II型周细胞显示分枝形态，其特征在于小细胞体和长而薄的过程（ 图2 ）。此时，大多数II型细胞周期仍保持为Nestin-GFP + ，而I型周细胞为Nestin-GFP（ 图2 ）。 另外，类型I，但不是类型II，pericy在脂肪形成培养基中14天后，tes分化成表达脂蛋白的脂肪细胞（ 图3 ）。 II型，但不是I型，在肌原基培养基中14天后，周细胞分化成表达S-myosin的肌管（ 图4 ）。这些结果强烈地表明I型周细胞是脂肪形成的，而II型周细胞是肌原性的。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55904/55904fig1.jpg" /> 图1 :门控边界和代表性排序。 （ A ）PDGFRβ-PE-FMO对照的荧光图，证明PDGFRβ-PE +巢蛋白-GFP和PDGFRβ-PE +巢蛋白-GFP +门控边界。 （ B ）显示PDGFRβ-PE + Nestin-GFP分布的样品的代表性荧光图<s（I型周细胞）和PDGFRβ-PE +巢蛋白-GFP + （II型周细胞）细胞。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55904/55904fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55904/55904fig2.jpg" /> 图2: 分类I型和II型周细胞中的形态和巢蛋白-GFP表达。将分选的I型和II型周细胞接种在盖玻片上并在周细胞培养基中生长3天。 I型周细胞显示圆形细胞体，具有短的过程，并且在荧光显微镜（激发激光:488nm;激发和发射滤光片:分别为470 / 40nm和515 / 30nm）下没有内源GFP信号。 II型细胞周期细胞表现出小细胞体，具有长而薄的过程，并具有强的内源GFP信号荧光显微镜在相同的设置下。比例尺= 100μm <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55904/55904fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55904/55904fig3.jpg" /> 图3: 分选的I型和II型周细胞的脂肪形成分化。分选的I型和II型周细胞在周细胞培养基中生长3天。然后将它们在脂肪形成培养基中分化14天。将细胞固定并用抗Perilipin抗体免疫染色，随后用Alexa555驴抗兔抗体进行免疫染色。免疫细胞化学显示，在荧光显微镜下（激发激光:543nm;激发和发射滤光片:540/45nm和600/50nm，周围的环境中，周细胞表达I型，但不是II型，表达脂肪细胞标记物perilipin（红色）<html>结构延续）。比例尺=50μm <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55904/55904fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/55904/55904fig4.jpg" /> 图4 :分选的I型和II型周细胞的肌原性分化。分选的I型和II型周细胞在周细胞培养基中生长3天，然后在肌生成培养基中分化14天。将细胞固定并用抗S-肌球蛋白抗体和Alexa555驴抗小鼠抗体免疫染色。免疫细胞化学显示，在荧光显微镜下（激发激光:543nm;激发和发射滤光片:540 / 45nm和600 / 50nm），周细胞表型II型，而不是I型周细胞表达成熟肌管/肌纤维标记S-肌球蛋白（红色） ， 分别）。刻度棒=50μm//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55904/55904fig4large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <table fo:keep-together.within-page="1"><tbody><tr><td> 管 </td><td> 染色 </td></tr><tr><td>无污染的控制</td><td>来自野生型小鼠的单细胞悬浮液</td></tr><tr><td> DAPI单色控制（死细胞排除） </td><td>来自野生型小鼠+ DAPI（5μg/ mL）的单细胞悬液</td></tr><tr><td> GFP单色控制</td><td>来自Nestin-GFP小鼠的单细胞悬浮液</td></tr><tr><td> PE单色控制</td><td> OneComp eBeads +PDGFRβ-PE抗体（4μg/ mL） </td></tr><tr><td> PE-FMO控制</td><td>来自Nestin-GFP小鼠+ DAPI（1μg/ mL）的单细胞悬液</td></tr><tr><td>样品</td><td>来自Nestin-GFP小鼠+PDGFRβ-PE抗体（4μg/ mL）的单细胞悬液+DAPI（1μg/ mL） </td></tr></tbody></table> 表1:单色对照，PDGFRβ-PE-FMO对照和肌肉样品的染色方案。

Watch this Scientific Journal Video about Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles at JoVE.com

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

这项工作描述了基于FACS的协议，允许从骨骼肌容易和同时地分离I型和II型周细胞。

从小鼠骨骼肌分离I型和II型周细胞

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

isolation-of-type-i-and-type-ii-pericytes-from-mouse-skeletal-muscles

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.1K 次观看.  University of Minnesota. 这项工作描述了基于FACS的协议，允许从骨骼肌容易和同时地分离I型和II型周细胞。 

视频: Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

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

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

隔离和卫星细胞的表征鼠头Branchiomeric肌肉

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

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

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

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

使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

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

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

新生小鼠胰腺胰岛功能的有效隔离

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

分离和新型表面标记组合来标识肝祖亚群富集

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

人诱导多能干细胞的前脑型脑 Organoids 标准化和可再生性的生成

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

嗜酸性粒植物视网膜免疫组织化学、西方分析和 Rna 分离的解剖

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

从骨骼肌活检程序分离、培养、表征和分化人类肌肉祖细胞

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

小鼠骨骼肌肉外泄原发性肌基器的分离和分化

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

翻译核糖体亲和纯化（TRAP）以研究细胞类型特异性尺度下阿拉伯生物基塔利亚纳根发育

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...