This work was supported by the European Research Council (ERG) and EMBO installation (ERG) and by a PhD fellowship from the Funda&#231;&#227;o para a Ci&#234;ncia e Tecnologia (MP).

注意:一个鼠标的产量足够的成肌细胞大约两张35毫米的菜肴或两个活成像菜，所以计划mattings，夹层，和涂层（步骤2.6）相应。 10只动物 - 由于肌细胞是通过连续的离心和预镀隔离，协议应在5个批次进行。 所有涉及动物对象的程序是由动物伦理委员会研究所德MEDICINA分子与大学皮埃尔与玛丽·居里批准 1.新生小鼠后肢肌肉解剖<ol><li>提前（ 材料表 ）准备所有的解决方案，并通过过滤（0.22微米过滤器）消毒。确保所有媒体都在37℃下除细胞之前，除了含有基底膜基质（ 例如，基质胶）的制剂。 </li><li>消毒夹层材料（每个之一:剪子弯，直剪刀，镊子定期，和细尖镊子），并通过用70％乙醇擦拭的工作台上。 </li><li>制备100毫米培养皿用5ml Dulbecco氏磷酸盐缓冲盐水（DPBS）的肌肉收集和保持在冰上，直到切碎步骤。 </li><li>斩首P6 - P8小鼠直剪刀和消毒用70％乙醇的皮肤。 </li><li>使背部皮肤切口，然后轻轻向外拉出后肢，直到它被删除，完全暴露后肢肌肉组织。 </li><li>使用钳去除脂肪组织而不损伤肌肉。 </li><li>要卸下背后肢肌肉，保持伸展肢体和弯曲的爪子，露出脚后跟肌腱。骨用剪子弯分离肌肉，从肌腱出发，通过轻轻滑动和切割以上。切除的肌肉，并放置在冰DPBS。 </li><li>由俭用细尖镊子肌肉和周围切割而不会损坏或股骨的膝关节隔离股四头肌。</li><li>解剖所有动物后，继续到无菌层流细胞培养罩，确定应执行以下所有步骤。 </li></ol> 2，成肌细胞分离<ol><li>除去多余的DPBS的。讳言灭菌弯曲剪刀组织，以获得一个均匀的质量。 </li><li>收集在用5毫升消化混合物的50毫升锥形离心管中的组织糜和在37℃搅拌孵育它为90分钟。 </li><li>通过在75 XG加入6毫升夹层介质和离心5分钟的悬浮液，以沉淀剩余的组织停止消化。 </li><li>仔细收集上清。 确保不收集组织碎片 。在350×g离心5分钟离心它;它悬浮在5毫升夹层介质中。 </li><li>通过40微米的细胞滤网过滤细胞悬浮液。添加25夹层介质中的溶液，并preplate它在150毫米的菜用于在细胞培养孵育箱（4小时37℃，5％CO 2的&lt;/子&gt;），以允许成纤维细胞附着。 </li><li>在冷的IMDM 100在RT 1小时:在预镀，外套菜肴基底膜基质的500微升稀释1。用DPBS洗一次，并立即板上的细胞（步骤2.8）或生长培养基，直到电镀离开。 </li><li>预镀后，收集上清液并在350 xg离心离心它为10分钟。 </li><li>重悬在它生长介质和细胞计数的血球。调节音量，使细胞150,000 250,000是每个基底膜基质涂，镀菜。保持所述细胞在细胞培养孵化器。 </li></ol> 3.肌纤维分化注:3天后，将细胞应在约70％汇合（ 图1B）开始保险丝和形式肌管。 <ol><li>在这点上，转染的细胞，如果需要的话，用siRNA或感兴趣的DNA。如果细胞不被转染，直接切换到分化培养基和滑雪p依次3.4。 </li><li>用按照制造商的说明书转染试剂转染。孵育用siRNA脂质复合物5小时的细胞（为20nM + 1微升试剂）或DNA-脂质复合物（1微克试剂+1μL）。如有必要，优化siRNA和DNA的浓度。 </li><li>与分化培养基清洗过一次，然后切换到新的分化培养基。 </li><li>在冰冷的分化培养基2:第二天，稀释基底膜基质1。删除现有的媒体，并添加冰冷的基质200微升到每道菜。 </li><li>孵育在细胞培养孵化30分钟。 </li><li>补充与集聚蛋白的分化培养基（100毫微克/毫升），并小心加入2毫升到细胞中。 </li><li>仔细改一半培养基的每2天，总是与集聚蛋白补充至100毫微克/微升的最终浓度。 </li><li>监测细胞分化和生存力。取决于多种因素（如胎牛血清和chicken胚胎提取物的起源），细胞可能需要5之间- 10分化d可达到完全成熟（ 图2）。 </li></ol> 4.免疫染色玻璃底菜<ol><li>对于免疫染色，在感兴趣的任何时间点，用DPBS洗涤细胞一次，并解决这些问题用4％PFA在RT 10分钟。 </li><li>用DPBS洗两次。在这一点上，可以将细胞储存于4℃。 </li><li>用0.5％的Triton X-100通透它们在RT 5分钟。 </li><li>用PBS洗两次，并用在室温阻断30分钟溶液阻塞。 </li><li>用在阻断溶液O / N在4℃下稀释的初级抗体孵育它们。 </li><li>洗3次用DPBS在RT 5分钟。 </li><li>与二级抗体和DAPI的0.2微克/毫升1小时在室温下孵育它们。 </li><li>洗3次用DPBS在RT 5分钟。 </li><li>添加安装介质的200μL并继续成像。 </li></ol>

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B., Wang, Z., Ross, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+mass+and+distribution+in+468+men+and+women+aged+18-88+yr.">Skeletal muscle mass and distribution in 468 men and women aged 18-88 yr.</a> J Appl Physiol. 89 (1), 81-88 (2000).</li><li>Manring, H., Abreu, E., Brotto, L., Weisleder, N., Brotto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+excitation-contraction+coupling+related+genes+reveal+aspects+of+muscle+weakness+beyond+atrophy-new+hopes+for+treatment+of+musculoskeletal+diseases.">Novel excitation-contraction coupling related genes reveal aspects of muscle weakness beyond atrophy-new hopes for treatment of musculoskeletal diseases.</a> Front Physiol. 5, 37 (2014).</li><li>Yaffe, D., Saxel, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+passaging+and+differentiation+of+myogenic+cells+isolated+from+dystrophic+mouse+muscle.">Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle.</a> Nature. 270 (5639), 725-727 (1977).</li><li>Pasut, A., Jones, A. E., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Culture+of+Individual+Myofibers+and+their+Satellite+Cells+from+Adult+Skeletal+Muscle.">Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle.</a> J Vis Exp. (73), (2013).</li><li>Meng, H., Janssen, P. M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Triage+and+Freezing+for+Models+of+Skeletal+Muscle+Disease.">Tissue Triage and Freezing for Models of Skeletal Muscle Disease.</a> J Vis Exp. (89), (2014).</li><li>Demonbreun, A. R., McNally, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Electroporation,+Isolation+and+Imaging+of+Myofibers.">DNA Electroporation, Isolation and Imaging of Myofibers.</a> J Vis Exp. (106), (2015).</li><li>Falcone, S., Roman, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-WASP+is+required+for+Amphiphysin-2/BIN1-dependent+nuclear+positioning+and+triad+organization+in+skeletal+muscle+and+is+involved+in+the+pathophysiology+of+centronuclear+myopathy.">N-WASP is required for Amphiphysin-2/BIN1-dependent nuclear positioning and triad organization in skeletal muscle and is involved in the pathophysiology of centronuclear myopathy.</a> EMBO Mol Med. 6 (11), 1455-1475 (2014).</li><li>Flucher, B. E., Phillips, J. L., Powell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dihydropyridine+receptor+alpha+subunits+in+normal+and+dysgenic+muscle+in+vitro:+expression+of+alpha+1+is+required+for+proper+targeting+and+distribution+of+alpha+2.">Dihydropyridine receptor alpha subunits in normal and dysgenic muscle in vitro: expression of alpha 1 is required for proper targeting and distribution of alpha 2.</a> J Cell Biol. 115 (5), 1345-1356 (1991).</li><li>Cooper, S. T., Maxwell, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C2C12+co-culture+on+a+fibroblast+substratum+enables+sustained+survival+of+contractile,+highly+differentiated+myotubes+with+peripheral+nuclei+and+adult+fast+myosin+expression.">C2C12 co-culture on a fibroblast substratum enables sustained survival of contractile, highly differentiated myotubes with peripheral nuclei and adult fast myosin expression.</a> Cell Motil Cytoskeleton. 58 (3), 200-211 (2004).</li><li>Al-Qusairi, L., Laporte, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-tubule+biogenesis+and+triad+formation+in+skeletal+muscle+and+implication+in+human+diseases.">T-tubule biogenesis and triad formation in skeletal muscle and implication in human diseases.</a> Skelet Muscle. 1, 26 (2011).</li><li>Vilmont, V., Cadot, B., Ouanounou, G., Gomes, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+to+study+mechanisms+of+neuromuscular+junction+development+and+maintenance.">A system to study mechanisms of neuromuscular junction development and maintenance.</a> Development. , (2016).</li><li>Vilmont, V., Cadot, B., Vezin, E., Le Grand, F., Gomes, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynein+disruption+perturbs+post-synaptic+components+and+contributes+to+impaired+MuSK+clustering+at+the+NMJ:+implication+in+ALS.">Dynein disruption perturbs post-synaptic components and contributes to impaired MuSK clustering at the NMJ: implication in ALS.</a> Sci Rep. 6, 27804 (2016).</li></ol>

作者什么都没有透露。

使用这种协议的主要成肌细胞的培养产生了一个特殊的利基，极大地培育肌纤维的发展。这部分是由于其也存在于非常小的数字的其它细胞类型。必须实现成肌细胞的浓度和纯度文化之间的平衡。一个良好的细胞培养物也取决于用于所述介质制剂中的产品的质量。动物来源的所有产品应彻底测试。在我们的经验中，消化条件下也应被监控。 像往常一样，小学文化，实验变异可能比孤立纤维或成肌细胞永生化的研究水平。这种变异可以通过介质和消化成分，小鼠的年龄和大小，以及用于培养操作和结果收集时间点的标准化减少。然而，在实时审议的错综复杂的机制的优点必要的肌纤维的发展大大超过了可变性的缺点。 这个协议赋予的体外方法的优点而不损害细胞的分化。肌纤维成熟直至形成打黑收缩偶联钙火花。这些功能性的输出可以在不同的实验条件下进行访问。此外，可存在到协议作出许多技术的变化。成肌细胞可以从与肌肉发育的兴趣突变新生小鼠收获。细胞可被裂解为在不同分化的时间点的生化分析。钙指标可以加到培养遵循其动态。光遗传学的结构可以用来执行一定的信号通路或诱导特异性当地反应。最后，肌纤维可以与其他细胞类型一起共培养，以研究它们的相互作用。

骨骼肌组成到体重1的40％。肌肉相关疾病代表一个巨大的健康和经济负担2。如何形成这种高度复杂的和有组织的组织，维持和再生的构成广泛和很好建立的研究领域。取决于具体的科学兴趣，最适合的方式的范围可以从简单的肌管培养物，以复杂的体内模型3 - 6。 该协议的目的是提供一种体外系统，该系统允许通过实时成像和免疫监视肌形成的。与传统方法相比，该系统提供了一个非常完整的和动态的洞察鼠标生肌过程。细胞可以从成肌细胞阶段横向显示三合会及周边核7成熟，多核肌纤维被遵循。这个成熟水平可使用常规细胞培养设备来实现，而不需要复杂的刺激或机械装置。虽然在体外系统一些成功的报道8,9，据我们所知，这是唯一的协议产生与T管与肌浆网（SR）横向配对鼠标成熟肌纤维。因此，该体外系统可以用来研究三元形成的分子机制，目前仍在知之甚少10。 使用该系统的进一步的优点是有效的鼠标定位资源，诸如抗体，药物，和RNAi工具的可用性。相对简单的协议不需要费力的步骤，技术娴熟的操作，或昂贵的和专用设备。成熟肌纤维开始5天的文化差异7后出现的，显示收缩加之钙火花（未公布数据）。在一个星期内，在不同的发展在哺乳动物体中最复杂的细胞之一的人阶段可以结合研究与各种体外测定法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dispase II</td>
			<td>Gibco</td>
			<td>17105041</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase&#160;type V</td>
			<td>Sigma-Aldrich-Aldrich</td>
			<td>C9263</td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM, Glutamax supplemented</td>
			<td>Gibco</td>
			<td>31980022</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Growth reduced factor</td>
			<td>Corning</td>
			<td>354230</td>
			<td>protein concentration of the lot should be around 10mg/ml and endotoxin result should be &#60;1.5</td>
		</tr>
		<tr>
			<td>Chicken Embryo Extract</td>
			<td>MP biomedical</td>
			<td>2850145</td>
			<td>it is also possible to prepare in the lab (Danoviz ME, Yablonka-Reuveni Z. Methods Mol Biol (2012))</td>
		</tr>
		<tr>
			<td>Recombinant rat agrin</td>
			<td>R&#38;D systems</td>
			<td>550-AG-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>GE Healthcare&#160;</td>
			<td>11581831</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX</td>
			<td>Invitrogen</td>
			<td>13778-150</td>
			<td>used to transfect siRNA</td>
		</tr>
		<tr>
			<td>Lipofectamine LTX</td>
			<td>Invitrogen</td>
			<td>15338-100</td>
			<td>used to transfect DNA</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668030</td>
			<td>used to transfect siRNA plus DNA</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190094&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Eurobio</td>
			<td>CVFSVF0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>Corning</td>
			<td>21008-949</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorodishes</td>
			<td>World precision instruments</td>
			<td>FD35-100</td>
			<td>dishes used to cultivate cells for live imaging</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>16% PFA (Paraformaldehyde)</td>
			<td>Science Services GmbH</td>
			<td>E15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Serum</td>
			<td>Sigma-Aldrich</td>
			<td>G9023</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA (Bovine Serum Albumine)</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponine</td>
			<td>Sigma-Aldrich</td>
			<td>47036</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>use at 200ng/ul</td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>SouthernBiotech</td>
			<td>0100-01&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Solutions and Media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digestion Mix</td>
			<td></td>
			<td></td>
			<td>in DPBS 
			5 mg/ml collagenase 
			3.5 mg/ml dispase 
			sterile filtered, can be stored in working aliquotes for 2 weeks at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Dissection Medium</td>
			<td></td>
			<td></td>
			<td>in IMDM Glutamax supplemented 
			10% FBS 
			1% Penicillin-Streptomycin 
			sterile filtered</td>
		</tr>
		<tr>
			<td>Growth Medium</td>
			<td></td>
			<td></td>
			<td>in IMDM Glutamax supplemented 
			20% FBS 
			1% Chicken Embryo Extract 
			1% Penicillin-Streptomycin1% Penicillin-Streptomycin 
			sterile filtered</td>
		</tr>
		<tr>
			<td>Differentiation Medium</td>
			<td></td>
			<td></td>
			<td>in IMDM Glutamax supplemented 
			2% Horse Serum 
			1% Penicillin-Streptomycin 
			sterile filtered</td>
		</tr>
		<tr>
			<td>Blocking Solution</td>
			<td></td>
			<td></td>
			<td>in DPBS 
			10% Goat Serum 
			5% BSA 
			add 0.1% saponine when incubating with primary and secondary antibodies</td>
		</tr>
	</tbody>
</table>

in vitro differentiation of mature myofibers for live imaging

骨骼肌是由肌纤维，在哺乳动物体内最大的细胞和少数的合胞体中的一个。如何，组成它的复杂性和进化上保守的结构组装仍在调查之中。它们的大小和生理特征往往限制操纵和成像应用。永生化细胞系的培养被广泛使用，但它只能复制分化的早期步骤。 在这里，我们描述了一个协议，使从鼠标主成肌细胞起源的肌纤维容易遗传操作。分化的一周后，将肌纤维显示收缩性，对齐肌节和三单元组，以及外围核。整个分化过程可以跟随实时成像或免疫荧光。该系统结合了现有离体和体外协议的优点。容易和有效转染的可能性，以及四通八达的交通网络的所有分化阶段扩大了潜在的应用。肌纤维随后可不仅用来解决相关发育和细胞生物学的问题，但也再现肌肉疾病表型为临床应用。

肌纤维发育的程度主要由隔离成肌细胞的纯度和可行性决定。的粘附，增殖和融合的能力可以用来凭经验访问这些参数（ 图1的A，B）。在增殖D2，成肌细胞应该坚持并应该显示典型的梭形。增殖预计在此阶段广泛地发生，导致自发肌管形成翌日（ 图1B）。 细胞汇合可能需要稍作调整。如果成肌细胞需要超过3天的增殖和熔断器它应该增加。如果肌纤维不能生长和细长相对直由于它们的密度应当减少。汇合典型地从中心向盘的外周降低，所以最好肌纤维应朝向外区域被发现。 肌管将快速伸长和显示多个中心对准的细胞核（ 图1C）。由D5，部分细胞开始采集条纹和移动他们的原子核外围。成熟特征的肌纤维的数量将随时间以及与单元厚度（ 图1D）增加。 分化程度可通过免疫荧光来进一步观察。固定在分化D8目前横向黑社会肌纤维。这可以通过T管（DPHR）和SR（triadin），预计在三单元组（ 图2）到共定位的成像元件来确认。 肌纤维的功能性可以通过实时成像来解决。从分化D3起，单元格显示自发抽搐。通过转染钙传感器（ 如:，GCaMP6f 11）中 ，有可能观察到收缩联接与钙的峰（ 图3）。 使用该系统，我们能够以确定在中央核肌病和肌强直性营养不良打乱一个新的分子途径，因此其可以是对创新分子疗法7的新靶点。我们也适于此方法研究了神经肌肉接头（NMJ）12的开发。通过与大鼠脊髓外植体的共培养，我们已在NMJ形成13描述了用于动力蛋白的作用。 <img alt="图1" src="/files/ftp_upload/55141/55141fig1.jpg" /> 图 1: 成肌细胞文化发展阶段。 A）在扩散D2，成肌细胞一直坚守并开始激增。 B）在prolif关合作D3，60铺满 - 80％达到和成肌细胞开始自发地融合。 c）在分化D3，包含位于中央的细胞核肌管占主导地位。 D）从分化D5起（ 例如8天），肌纤维开始呈现出条纹和外设核并开始变厚。比例尺:50微米。 <a href="http://ecsource.jove.com/files/ftp_upload/55141/55141fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55141/55141fig2.jpg" /> 图2:D8肌纤维免疫显的代表共焦成像。 A）免疫染色二氢吡啶受体（DHPR，上图）和triadin（TRDN，中间面板）。在DHPR，TRDN和DAPI通道的重叠图显示了黑社会组件共存。 B）<html>在答Ç绘制的黄线）染色α辅肌动蛋白（绿色）和DAPI（蓝色）肌纤维的容积再现的三维图像的强度分布。比例尺和网格宽度:5微米。 <a href="http://ecsource.jove.com/files/ftp_upload/55141/55141fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55141/55141fig3.jpg" /> 图 3: 自发性抽搐肌纤维钙水平的实时成像。在抽动肌纤维钙火花的 ）高速长延时（20ms的帧）显微镜。通过GCaMP6f的表达（Addgene质粒＃40755）中检测到的钙。 B）的荧光强度的定量随时间在面板A中的钙传感器_blank"&gt;请点击此处查看该图的放大版本。

Watch this Scientific Journal Video about In Vitro Differentiation of Mature Myofibers for Live Imaging at JoVE.com

In Vitro Differentiation of Mature Myofibers for Live Imaging

Muscle cells are among the most complex eukaryotic cells. We present a protocol for the in vitro differentiation of highly mature myofibers that allows for genetic manipulation and clear imaging during all developmental stages.

在成熟的肌纤维诱导分化实时成像

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily ...

in-vitro-differentiation-of-mature-myofibers-for-live-imaging

Research

JoVE Journal

Developmental Biology

In Vitro Differentiation of Mature Myofibers for Live Imaging

10.2K 次观看.  Universidade de Lisboa. Muscle cells are among the most complex eukaryotic cells. We present a protocol for the in vitro differentiation of highly mature myofibers that allows for genetic manipulation and clear imaging during all developmental stages.

视频: In Vitro Differentiation of Mature Myofibers for Live Imaging

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

实时成像<em&gt;果蝇</em&gt;蛹眼

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

科研

发育生物学

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

住发展拟南芥花的共焦成像

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

在人多能的体外干细胞向滋养层细胞

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

长期的实时成像<em&gt;果蝇</em&gt;眼睛光盘

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

有丝分裂过程中染色体分离的活体细胞成像

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic development. The activity and morphology of SAMs determine important agronomic traits, such as shoot architecture, size and number of reproductive organs, and most importantly, grain yield. Here, we provide a detailed protocol for analyzing both the surface morphology and the internal cellular structure of the living SAMs from different species through laser scanning confocal microscope. The whole procedure from the sample preparation to the acquisition of high resolution three-dimensional (3D) images can be accomplished within as short as 20 minutes. We demonstrate that this protocol is highly efficient for studying not only the inflorescence SAMs of the model species but also the vegetative meristems from different crops, providing a simple but powerful tool to study the organization and development of meristems across different plant species.

This protocol presents how to live image and analyze the shoot apical meristems from different plant species using laser scanning confocal microscopy.

不同植物种的射击顶端共聚焦活图

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

果蝇胚胎肌肉收缩的实时成像和分析

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

在成熟的肌纤维诱导分化实时成像

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