Name: isolation of human myoblasts, assessment of myogenic differentiation, and store-operated calcium entry measurement
Uploaded: 2017-07-26T12:30:00.000+00:00
Duration: 10 min 45 s
Description: Watch this Scientific Journal Video about Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement at JoVE.com

这项工作得到了瑞士国家科学基金会（拨款号310030-166313），"基金会马塞尔·莱瓦兰特基金会"以及"基金会报告"的支持。

在矫形外科手术中收集人体肌肉样本（从半神经肌肉获得的组织）作为外科手术废物。与人类研究有关的所有方法都是按照瑞士监管机构的指导方针和条例进行的，并经瑞士日内瓦省政府批准的瑞士法典委员会批准（议定书CER号12-259） 。从参与研究的所有成年受试者获得知情和书面同意。 1.从人类骨骼肌分离成肌细胞<ol><li>在所有程序中，通过在II级组织培养罩下工作并使用无菌培养基和无菌缓冲液来维持无菌条件。 注意:此过程可应用于许多人类骨骼肌肉。 </li></ol>解离协议<ol><li>将肌肉样品（2-3克）转移到无菌6厘米盘子。 </li><li>用5-10毫升磷酸盐缓冲盐水（PBS）洗涤肌肉样品。重复洗涤。 </li><li>使用弯曲的剪刀和夹具去除结缔组织和脂肪组织。 </li><li>称重肌肉样品（参见步骤2.13），并将其转移到新的无菌6厘米平板上。 </li><li>加入5 mL的0.05％胰蛋白酶-EDTA。 </li><li>使用剪刀，将肌肉组织切成小块（小于2毫米）。 </li><li>使用10mL血清移液管将切碎的肌肉转移到含有磁条的200mL解离瓶中。重复此步骤，直到将90mL的0.05％胰蛋白酶-EDTA与所有切碎的肌肉一起转移至解离盒。关闭解离瓶，并在温和搅拌下在37℃下孵育60分钟。 </li><li>从加热浴中取出解离瓶，并通过向解离瓶中加入10mL胎牛血清（FCS; 10％最终体积）停止酶反应。用移液管均质化混合物。 </li><li>预备是两个50 mL管，每个具有70μm的细胞过滤器。通过在过滤器上上下移液直到悬浮液通过，将每个管上的细胞过滤器的一半通过细胞过滤器。 </li><li>将管子在200xg和20℃下离心5分钟;吸出并丢弃上清液。 </li><li>用10ml生长培养基（GM; 表1 ）重悬细胞，并将细胞通过置于一个50mL管上的40-μm细胞过滤器。将管子在200xg和20℃下离心5分钟;吸出并丢弃上清液。 </li><li>用10 mL GM悬浮细胞，并以200 xg和20℃离心管5 min;吸出并丢弃上清液。 </li><li>用1mL GM重悬细胞，并使用Neubauer室计数细胞。 注意:通常，每克肌肉的产量应为3×10 5个细胞。通过将细胞数除以肌肉生物体的重量来计算产量sy（步骤2.4）。 </li><li>将2×10 5个细胞置于含有4mL GM的无菌6cm平板中，并在37℃和7％CO 2下孵育。根据需要准备尽可能多的板材。 </li><li> 3天后更改GM。 注意:新鲜分离的细胞需要时间粘附到培养板上。此外，第一次造血细胞倍增时间约为50-60小时。第一师后，倍增时间约为20小时。 </li><li> 5 - 7天后，当细胞达到70-80％融合（约1.5×10 6个细胞/每6cm平板）时，开始抗体染色和细胞分选（步骤3）。 </li></ol> 3.抗体染色和细胞分选<ol><li>用PBS冲洗一次后，用37℃和7％CO 2的 1mL 0.05％胰蛋白酶-EDTA将贴壁细胞（6cm平板）孵育3分钟（或以5％CO 2孵育）。通过加入2mL GM停止反应，并将细胞收集在15mL管中。离心（200×g 5分钟）后，在1 mL的GM中通入细胞。 </li><li>使用Neubauer室计数细胞。 </li><li>在冰上执行以下步骤。 注意:在细胞计数器上设置正确的荧光补偿的程序是必不可少的，任何细胞仪都应该相似。管1和管2用作阴性对照。管3-7用于在第一次实验期间在血细胞仪上设置补偿（ 表2 ）。用于每种抗体及其相应同种型的最终浓度应相同（约1μg/ 10 6个细胞）。 </li><li>对于管1-7，分配1×10 5个细胞/管（5-mL管）（ 表2 ）。将剩余的细胞悬浮液放在管8中进行细胞分选。 </li><li>用1 mL冷FACS缓冲液（PBS-5％FCS）清洗细胞。关闭并在200 xg和4℃离心管5分钟;吸出并丢弃上清液。 </li><li>每管加入200μLFACS缓冲液。加入抗体e管根据表2 。在冰上孵育30分钟。 </li><li>洗涤细胞:向每个管中加入1 mL FACS缓冲液。关闭并以200 xg和4℃离心管5 min。吸出并丢弃上清液。再一次重复这一步。 </li><li>将细胞重悬在500μL的FACS缓冲液中。关闭管子并将其保持在冰上直到细胞分选。准备4管1.5 mL，每个管含有500μL的GM，在细胞分选过程中收集细胞是必需的。把它们放在冰上。 </li><li>通过流式细胞术分析人类成肌细胞（ 图1 ）。排除CD45阳性造血细胞后，基于CD56表达分离CD45阴性细胞。然后，将CD34，CD144和CD146（人成肌细胞定义为CD56 + / CD146 + / CD45- / CD34- / CD144-细胞）的CD56阳性细胞门。重新分析一小部分排序的细胞群以检查纯度。 </li><li>将含有人类myobla的管放在一起sts在一个15-mL管中，并通过加入5mL GM洗涤一次。将管子在200xg和20℃下离心5分钟;吸出并丢弃上清液。 </li><li>将细胞重悬于1mL GM中，并在含有4mL GM（2×10 5个细胞/平板）的未涂覆的6cm平板上平板培养细胞。在37℃和7％CO 2下孵育。 </li></ol> 4.细胞维护<ol><li>每2天更换一半的培养基。从培养皿中取出2 mL老GM，加入2 mL GM。 注意:成肌细胞产生有利于培养条件的生长因子。 </li><li>当细胞达到70-80％汇合时，对细胞进行胰蛋白酶化，以避免预分化。用PBS冲洗一次后，用37℃和7％CO 2的 1mL 0.05％胰蛋白酶-EDTA将贴壁细胞（6cm平板）孵育3分钟。通过加入2mL GM停止反应并将细胞收集在15mL管中。离心（200×g，5分钟）后，将细胞悬浮在1 mL GM中。 </li><li>计数细胞并准备​​6cm的平板，每个板2×10 5个细胞在4mL的GM中。 </li><li>保持人类成肌细胞达6代，或在含有10％DMSO的GM中冷冻，每毫升1×10 6个细胞。在逐层冷冻箱中冷冻细胞（1°C / min）;为了长期储存，将细胞置于液氮中。 </li></ol> 5.用siRNA转染人成肌细胞注意:在诱导分化前两天，使用商业转染试剂转染成肌细胞。特定siRNA对基因的作用总是与对照siRNA的作用进行比较。 siRNA是双链RNA，必须保存在冰上并用手套操作。 <ol><li>在6厘米平板（约1.5×10 6个细胞）中获得70-80％汇合的人成肌细胞单层。 注意:每次转染需要5×10 5个细胞以获得一个对照GM中2天后转染成肌细胞的流动单层。 </li><li>准备玻璃盖玻片（在步骤7中描述为钙成像）。 </li><li>热GM，0.05％胰蛋白酶-EDTA，PBS至37℃。 </li><li>在II级组织培养罩中执行以下所有步骤以维持无菌性。 </li><li>在1.5 mL试管中，在20μM下调出500μL血清培养基，3μL转染试剂和1μLsiRNA。轻轻混合并保持在室温（RT）15 - 20分钟。 </li><li>在孵育期间，准备成肌细胞进行转染。用PBS冲洗一次后，用37℃和7％CO 2的 1mL 0.05％胰蛋白酶-EDTA将贴壁细胞（6cm平板）孵育3分钟。通过加入2mL GM停止反应并将细胞收集在15mL管中。离心（200×g 5分钟）后，将细胞重悬于1mL的GM中。 </li><li>使用Neubauer室计数细胞。 </li><li>重悬5×10 5个细胞在每次转染的200μL的GM中（ 即，用于2次转染，重悬于400μL）。 </li><li>向每个500μLsiRNA转染混合物中加入200μL细胞悬浮液。上下移动两次，在室温下孵育5分钟。 </li><li>将700μL（细胞+ siRNA转染子）加入到含有玻璃盖玻片的3.5cm培养皿中。加入GM，终体积为2 mL。 </li><li>在37℃和7％CO 2下24小时后，用2mL新鲜GM完全替代培养基。另外24小时后，通常存在转染细胞的汇合单层;如果是这样，通过用2mL分化培养基（DM; 表1 ）代替GM来诱导成肌细胞分化。 </li><li>诱导分化后24 - 48 h或增殖成肌细胞进行钙成像实验。 注意:诱导分化后48 h通常进行免疫荧光。 </li></ol> Imm分化标记的非荧光染色注意:在DM中48小时后进行免疫荧光染色，但也可以在其他分化时间进行。肌细胞生成素和MEF2是仅在分化细胞中表达的核蛋白（融合成肌管之前），其染色允许评估分化过程。肌球蛋白重链（MyHC）仅在肌管中表达，用于估计肌管大小和融合指数（肌管数量/核总数）。 <ol><li>在DM中48小时后，在室温下用1mL PBS洗涤分化细胞的汇合单层两次。仔细做这个，以避免分离肌管。 </li><li>在室温下用1mL的4％多聚甲醛（PFA）将细胞固定15分钟。用PBS洗涤细胞两次，每次5分钟。 </li><li>用含有0.25％Triton X-100的1mL PBS将细胞渗透45分钟，并用1mL PBS洗涤3次。 </li><li>阻止不了通过在补充有0.2％Tween-10和2％山羊血清（阻滞溶液）的PBS中的细胞在室温下孵育30分钟来实现特异性结合位点。 </li><li>制备在块溶液中稀释的一抗（600μL/ 3.5cm板）。 注意:使用以下一抗:小鼠抗肌钙蛋白（1:600），兔抗MEF2（1:300）和小鼠抗MyHC抗体（MF20,1:1,000）。可以将相同的阻断溶液，小鼠抗肌球蛋白重链或小鼠抗肌发生蛋白与兔抗MEF2混合。 </li><li>去除阻滞溶液，每3.5厘米平板加入600μL一抗溶液，并在4℃下在加湿室中孵育过夜。 </li><li>用1 mL PBS洗涤细胞3次。山羊抗小鼠Alexa 488（1:1,000），山羊抗兔亚型Alexa 546（1:1,000），山羊抗小鼠Alexa 488（1:1000），在室温下孵育1小时，在室内600μL二嵌段溶液中制备的二抗，和DAPI（1:50，15μM的储备溶液）。 CAUTION:DAPI是用手套处理的有毒化合物。 </li><li>用1mL PBS吸出并洗涤细胞3次。 </li><li>使用具有防紫外线溶液的聚乙烯醇安装介质安装玻璃盖玻片。将它们保持在4°C，防止光照直到成像细胞。 </li></ol> 7.钙成像<ol><li>预先培养，准备直径25毫米的玻璃盖玻片。将盖玻片放在70％乙醇中48小时，以清除表面上的任何油脂和污垢，从而减少细胞粘附在盖玻片上。 </li><li>用水冲洗盖玻片几次后，将其放回水中并灭菌（高压灭菌）。 </li><li>每3.5厘米培养板放一块盖玻片，让它们干燥。储存培养皿进行进一步实验或立即使用。 </li><li>将转染的成肌细胞（5 - 6×10 5个细胞）加入到玻璃盖玻片中。 注意:诱导后24 - 48 h进行实验或在增殖成肌细胞上。 </li><li>为了进行Ca 2+成像，用PBS或直接用实验培养基（含钙培养基; 表3 ）清洗细胞2次。用2μMFura-2 / AM加上1μMPluronic酸将细胞在室温下在黑暗中加载约30分钟。 注意:Pluronic acid有助于溶解Fura-2。 Fura-2 / AM和pluronic acid溶解在含钙培养基中。 </li><li>在相同的培养基中洗涤细胞两次，但没有Fura-2 / AM和普朗尼克酸，并将其留在黑暗中另外10-15分钟以允许Fura-2 / AM的去酯化。 </li><li>如果Fura-2的加载效率太低，可以添加更多的Fura-2（ 例如 4μM）和/或将细胞孵育较长时间（ 例如 40分钟）。 </li><li>从培养皿中取出盖玻片并将其安装在实验室中。加入〜1 mL含钙培养基（取决于实验的体积精神室）。将其安装在显微镜舞台上并开始录制。 注意:荧光Ca 2+敏感探针Fura-2在340 nm和380 nm交替激发，发射光在510 nm处收集。随着Ca 2+浓度的波动相对较慢，每2秒获得1个比例。 </li><li> 为了激活SOCE，使用以下典型的沙星烟碱/ Ca 2+再添加方案:在含钙培养基中将细胞离开2-3分钟。用无钙培养基更换培养基1-2分钟。 <ol><li>加入1μP毒胡萝卜素消耗内部钙储存。 注意:Thapsigargin是肌质内质网Ca 2+ ATP酶（SERCA）泵活性的不可逆阻断剂.8-10分钟后，用含钙培养基快速置换培养基。 Ca 2+将通过SOCE通道进入细胞。等待约5分钟，然后终止实验。 小心:Thapsigargin是一种有毒的化合物，应用手套处理。 </li></ol></li></ol> 8. Ca 2+测量分析注意:使用采集软件进行分析。 <ol><li>打开实验。在没有细胞（背景区域）的区域中绘制区域1，并绘制要分析的细胞的细胞质中的其他区域（该区域可以在任何图像上绘制:340nm，380nm，或比例图像）。转到"运行实验"&gt;"参考图像"。在340和380频道下，选择"区域1"，然后点击"减去背景"。 </li><li>运行整个实验（F4:向前），以确保没有干扰背景区域，例如穿过背景区域的荧光灰尘斑点，并且在实验期间细胞不移动。 注意:所选择的兴趣区域不应包括黑暗地区;否则，比率值将显得嘈杂，因为背景的一部分将被包括在测量中。 </li><li>点击"日志数据"，再次播放整个实验。 注意:这将打开一个电子表格，其中记录时间和比率。还可以节省340 nm和380 nm的波长。 </li><li>要获得关于SOCE的信息，从实验中提取两个重要参数:Ca 2+再加入的幅度和Ca 2+进入阶段的斜率（ 图3 ）。 </li><li>通过从Ca 2+升高的最大幅度减去Ca 2+再添加之前的比值获得振幅。通过拟合Ca 2+重添加后第一秒的线性回归获得最大斜率。 注意:毒胡萝卜素反应曲线下的面积也是提取物的参考参数t，因为它反映了存储在SR中的游离钙的量。 </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+receptor+potential+cation+channels+in+disease.">Transient receptor potential cation channels in disease.</a> Physiol Rev. 87 (1), 165-217 (2007).</li><li>Vandebrouck, C., Martin, D., Colson-Van Schoor, M., Debaix, H., Gailly, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+TRPC+in+the+abnormal+calcium+influx+observed+in+dystrophic+(mdx)+mouse+skeletal+muscle+fibers.">Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers.</a> J Cell Bio. 158 (6), 1089-1096 (2002).</li><li>Antigny, F., Koenig, S., Bernheim, L., Frieden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=During+post-natal+human+myogenesis,+normal+myotube+size+requires+TRPC1-+and+TRPC4-mediated+Ca(2)(+)+entry.">During post-natal human myogenesis, normal myotube size requires TRPC1- and TRPC4-mediated Ca(2)(+) entry.</a> J Cell Sci. 126 (11), 2525-2533 (2013).</li><li>Brinkmeier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRP+channels+in+skeletal+muscle:+gene+expression,+function+and+implications+for+disease.">TRP channels in skeletal muscle: gene expression, function and implications for disease.</a> Adv Exp Med Biol. 704, 749-758 (2011).</li><li>Pan, Z., Brotto, M., Ma, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Store-operated+Ca2++entry+in+muscle+physiology+and+diseases.">Store-operated Ca2+ entry in muscle physiology and diseases.</a> BMB Rep. 47 (2), 69-79 (2014).</li><li>Lacruz, R. S., Feske, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diseases+caused+by+mutations+in+ORAI1+and+STIM1.">Diseases caused by mutations in ORAI1 and STIM1.</a> Ann N Y Acad Sci. 1356, 45-79 (2015).</li><li>Li, T., 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=STIM1-Ca(2+)+signaling+is+required+for+the+hypertrophic+growth+of+skeletal+muscle+in+mice.">STIM1-Ca(2+) signaling is required for the hypertrophic growth of skeletal muscle in mice.</a> Mol Cell Biol. 32 (15), 3009-3017 (2012).</li><li>Wei-Lapierre, L., Carrell, E. M., Boncompagni, S., Protasi, F., Dirksen, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orai1-dependent+calcium+entry+promotes+skeletal+muscle+growth+and+limits+fatigue.">Orai1-dependent calcium entry promotes skeletal muscle growth and limits fatigue.</a> Nat Commun. 4, 2805 (2013).</li><li>Konig, S., 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=Membrane+hyperpolarization+triggers+myogenin+and+myocyte+enhancer+factor-2+expression+during+human+myoblast+differentiation.">Membrane hyperpolarization triggers myogenin and myocyte enhancer factor-2 expression during human myoblast differentiation.</a> J Biol Chem. 279 (27), 28187-28196 (2004).</li><li>Crisan, M., 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=A+perivascular+origin+for+mesenchymal+stem+cells+in+multiple+human+organs.">A perivascular origin for mesenchymal stem cells in multiple human organs.</a> Cell Stem Cell. 3 (3), 301-313 (2008).</li><li>Bigot, A., 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=Age-Associated+Methylation+Suppresses+SPRY1,+Leading+to+a+Failure+of+Re-quiescence+and+Loss+of+the+Reserve+Stem+Cell+Pool+in+Elderly+Muscle.">Age-Associated Methylation Suppresses SPRY1, Leading to a Failure of Re-quiescence and Loss of the Reserve Stem Cell Pool in Elderly Muscle.</a> Cell Rep. 13 (6), 1172-1182 (2015).</li><li>Bettadapur, A., 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=Prolonged+Culture+of+Aligned+Skeletal+Myotubes+on+Micromolded+Gelatin+Hydrogels.">Prolonged Culture of Aligned Skeletal Myotubes on Micromolded Gelatin Hydrogels.</a> Sci Rep. 6, 28855 (2016).</li><li>Darbellay, B., Arnaudeau, S., Bader, C. R., Konig, S., Bernheim, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STIM1L+is+a+new+actin-binding+splice+variant+involved+in+fast+repetitive+Ca2++release.">STIM1L is a new actin-binding splice variant involved in fast repetitive Ca2+ release.</a> J Cell Biol. 194 (2), 335-346 (2011).</li><li>Sauc, S., 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=STIM1L+traps+and+gates+Orai1+channels+without+remodeling+the+cortical+ER.">STIM1L traps and gates Orai1 channels without remodeling the cortical ER.</a> J Cell Sci. 128 (8), 1568-1579 (2015).</li><li>Demaurex, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+measurements+in+organelles+with+Ca2+-sensitive+fluorescent+proteins.">Calcium measurements in organelles with Ca2+-sensitive fluorescent proteins.</a> Cell Calcium. 38 (3-4), 213-222 (2005).</li></ol>

作者没有竞争的经济利益。

来自成年骨骼肌的人成肌细胞的分离和培养提供了体外模型来研究肌肉分化和肌肉再生。在本文中，我们提供了一种协议，允许以简单且成本有限的方式纯化高产量的人类成肌细胞。此外，该技术在分离的成肌细胞的百分比及其肌原性分化效率方面提供了可靠和可重现的结果。事实上，我们在细胞分选后获得约60％的人类成肌细胞。这些细胞可保持培养多达6代（约3...

人骨骼肌由肌原纤维前体细胞融合产生的收缩性多核肌肉纤维组成。由于肌肉纤维（肌膜）和基底层之间的骨骼肌干细胞存在于骨骼肌干细胞之间，骨骼肌具有在损伤后再生的能力。在未受伤的肌肉中，SC主要存在于静止状态。响应于机械应力或损伤，雪旺成为激活（成肌细胞），增殖，和经历或者分化以形成新的肌纤维或自我更新来补充SC池1，2。多年来，开发了几种技术来分离SCs及其后代，成肌细胞，从骨骼肌活组织检查。对这些细胞上表达的细胞表面标志物的更多了解和荧光激活细胞分选（FACS）的方法3，4，5，现在有可能对人成肌细胞的纯群体从肌肉样品隔离。 钙信号调节骨骼肌发育，体内平衡和再生。尤其是钙条目是继细胞内储存消耗激活，叫SOCE，是这些过程很重要6。在细胞刺激后，内/肌质网（ER / SR）中的Ca 2+水平降低，这反过来激活允许Ca 2+进入以重新填充ER / SR 7的质膜Ca 2+通道。 SOCE中的两种主要蛋白质是ER / SR Ca 2+ -感染基质相互作用分子1（STIM1）蛋白和质膜Ca 2+通道Orai1。骨骼肌丰富地表达了这两种蛋白质，以及同一家族的其他蛋白质（STIM2 aND Orai2-Orai3）8,9和几个的Ca 2+瞬时受体电位规范（TRPC）家族10，11，12，13的可透过的通道。通过SOCE通路的Ca 2+进入对肌肉形成/再生十分重要14 。 STIM1或Orai1蛋白的突变对肌肉功能的有害影响，主要导致肌肉张力减退15。与SOCE障碍动物模型也具有增强的易疲劳6，16，17显示降低的肌肉质量和力量，在一起。如上所述，其他STIM和Orai蛋白以及许多TRPC通道在骨骼肌中表达，并且它们各自的作用迄今尚未确定。敲门拥有自己的表达水平，因此可以调查他们对SOCE及其在人类骨骼肌分化过程中的作用的影响。 可以使用两种不同的方法进行SOCE测量:电生理电流记录和Ca 2+测量。电生理学肯定是一种更直接的方法，因为它测量了目前的兴趣及其相关的电生理特征。然而，这种技术很难应用于肌肉细胞，主要是因为肌肉细胞的大尺寸和内源性SOCE电流的小尺寸。细胞溶质Ca 2+成像在技术上是非常易于使用的，但测量更为间接，因为细胞质中测量的Ca 2+水平反映了Ca 2+的进入和再次抽出细胞或内部商店。 一种方法，包括从肌肉的小块分离成肌细胞本文解释了酶消化，扩增和FACS后的等信号。肌肉分化和免疫荧光协议遵循分化标志物的表达随时间的过程中描述18。最后，解释了SOCE的测量和不同离子通道在Ca 2+信号和骨骼肌分化中的作用。

<table><tbody><tr><td>Ham&amp;rsquo;s F10</td><td>ThermoFisher Scientific</td><td>31550-023</td><td></td></tr><tr><td>FCS</td><td>ThermoFisher Scientific</td><td>10270-106</td><td>Lot 41G5532K</td></tr><tr><td>BSA</td><td>Sigma</td><td>O5482</td><td></td></tr><tr><td>fetuin</td><td>Sigma</td><td>F3385</td><td></td></tr><tr><td>EGF</td><td>Corning</td><td>354001</td><td></td></tr><tr><td>Dexamethansone</td><td>Sigma</td><td>D1756</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>I9278</td><td></td></tr><tr><td>Creatine</td><td>Sigma</td><td>27900</td><td></td></tr><tr><td>Pyruvate</td><td>Sigma</td><td>P4562</td><td></td></tr><tr><td>Uridine</td><td>Sigma</td><td>U3003</td><td></td></tr><tr><td>Gentamycin</td><td>ThermoFisher Scientific</td><td>15710-049</td><td></td></tr><tr><td>Trypsin-EDTA 0.05%</td><td>ThermoFisher Scientific</td><td>25300-062</td><td></td></tr><tr><td>70 &amp;mu;m cell strainer</td><td>Falcon</td><td>352350</td><td></td></tr><tr><td>40 &amp;mu;m cell strainer</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>Falcon 50&amp;nbsp;mL tube</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Falcon 15&amp;nbsp;mL tube</td><td>Falcon</td><td>352096</td><td></td></tr><tr><td>Falcon 5&amp;nbsp;mL tube (FACS)</td><td>Falcon</td><td>352058</td><td></td></tr><tr><td>Culture medium: OPTI-MEM</td><td>ThermoFisher Scientific</td><td>31985-047</td><td></td></tr><tr><td>Transfectant reagnt: RNAiMax</td><td>ThermoFisher Scientific</td><td>1756064</td><td></td></tr><tr><td>PBS</td><td>ThermoFisher Scientific</td><td>14190-094</td><td></td></tr><tr><td>Mounting medium</td><td>Fluka</td><td>10981</td><td></td></tr><tr><td>Mouse anti-MyHC *</td><td>DSHB</td><td>MF20&amp;nbsp;</td><td>concentrate</td></tr><tr><td>Mouse anti-myogenin</td><td>BD Pharmigen</td><td>556358</td><td></td></tr><tr><td>Rabbit anti-MEF2</td><td>Santa Cruz Biotech</td><td>C-21</td><td></td></tr><tr><td>Goat anti-mouse Alexa488</td><td>ThermoFisher Scientific</td><td>A11029</td><td></td></tr><tr><td>Goat anti-rabbit Alexa 546</td><td>ThermoFisher Scientific</td><td>A11035</td><td></td></tr><tr><td>Mouse anti human CD56-Alexa488</td><td>BD Pharmingen</td><td>557699</td><td></td></tr><tr><td>Mouse anti human CD146-PECy7</td><td>BD Pharmingen</td><td>562135</td><td></td></tr><tr><td>Mouse anti human CD45-PE-CF594</td><td>BD Pharmingen</td><td>562279</td><td></td></tr><tr><td>Mouse anti human CD34-APC</td><td>BD Pharmingen</td><td>345804</td><td></td></tr><tr><td>Mouse anti human CD144-PE</td><td>BD Pharmingen</td><td>560410</td><td></td></tr><tr><td>Alexa Fluor 488 mouse IgG1k</td><td>BD Pharmingen</td><td>557702</td><td></td></tr><tr><td>PECy7 mouse IgG1k</td><td>BD Pharmingen</td><td>557872</td><td></td></tr><tr><td>PE-CF594 mouse IgG1k</td><td>BD Pharmingen</td><td>562292</td><td></td></tr><tr><td>APC mouse IgG1k</td><td>BD Pharmingen</td><td>550854</td><td></td></tr><tr><td>PE mouse IgG1k</td><td>BD Pharmingen</td><td>556650</td><td></td></tr><tr><td>DAPI</td><td>Sigma</td><td>D9542</td><td>CAUTION:&amp;nbsp; toxic compound</td></tr><tr><td>Paraformaldehyde</td><td>Fluka</td><td>762400</td><td></td></tr><tr><td>Fura-2 AM</td><td>ThermoFisher Scientific</td><td>F1201</td><td></td></tr><tr><td>Pluronic F-127</td><td>ThermoFisher Scientific</td><td>P3000MP</td><td></td></tr><tr><td>Thapsigargin</td><td>Sigma</td><td>T9033</td><td>CAUTION&amp;nbsp;: toxic compound&amp;nbsp;</td></tr><tr><td>Ratio imaging acquisition software: Metafluor 6.3</td><td>Molecular Device</td><td></td><td></td></tr><tr><td>*&amp;nbsp;: MF 20 was deposited to the DSHB by Fischman, D.A.&amp;nbsp;</td><td></td><td></td><td></td></tr></tbody></table>

isolation of human myoblasts, assessment of myogenic differentiation, and store-operated calcium entry measurement

卫星细胞（SC）是位于肌纤维质膜与周围基底层之间的肌肉干细胞。它们对于肌肉再生至关重要。在骨骼肌经常发生的损伤中，SC被激活。它们作为成肌细胞增殖并分化以修复肌肉损伤。在肌肉分化过程中发生的许多事件中，胞质Ca 2+信号是非常重要的。这些Ca 2+信号来自内部Ca 2+储存物的Ca 2+释放，以及来自细胞外空间的Ca 2+进入，特别是存储操作的Ca 2+进入（SOCE）。本文介绍了一种用于从矫形外科手术后收集的肌肉样品中获得纯人类成肌细胞群体的方法。机械和酶消化组织，扩增细胞，然后根据特异性的存在通过流式细胞术分选fic膜标记。一旦获得，通过从培养基中除去生长因子，使人成肌细胞扩大并致力于分化。特异性转录因子和体外免疫荧光的表达水平用于评估控制条件下和沉默参与Ca 2+信号传导的蛋白质后的肌原性分化过程。最后，我们详细介绍了使用Fura-2作为比较的Ca 2+探针，提供可靠和可重复的SOCE测量。

在人肌肉样品的酶解后，在GM中扩增细胞。在FACS后获得定义为CD56 + / CD146 + / CD45- / CD34- / CD144-细胞的人成肌细胞。成肌细胞占分析人口的60％以上（ 图1 ）。将原代人成肌细胞复制，生长至汇合，并在DM中培养48小时。免疫染色用于转录因子MEF2和肌肉特异性蛋白质肌球蛋白重链（MyHC）的表达，我们观察到大部分细胞在体外形成肌管（MyHC阳性细胞），融合指数为60.9±1.3?...

Watch this Scientific Journal Video about Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement at JoVE.com

Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement

在这里，我们描述了一种从成年肌肉组织中获得纯种人成肌细胞的方法。这些细胞用于研究体外骨骼肌分化，特别是研究参与Ca 2+信号传导的蛋白质。

人成肌细胞的分离，造血分化的评估和存储操作的钙进入测量

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for ...

isolation-human-myoblasts-assessment-myogenic-differentiation-store

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

10.0K 次观看.  Geneva University Hospitals & Faculty of Medicine. 在这里，我们描述了一种从成年肌肉组织中获得纯种人成肌细胞的方法。这些细胞用于研究体外骨骼肌分化，特别是研究参与Ca 2+信号传导的蛋白质。 

视频: Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement

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

In Vitro Differentiation of Mature Myofibers for Live Imaging

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily conserved structures that compose it are assembled remains under investigation. Their size and physiological features often constrain manipulation and imaging applications. The culture of immortalized cell lines is widely used, but it can only replicate the early steps of differentiation.
Here, we describe a protocol that enables easy genetic manipulation of myofibers originating from primary mouse myoblasts. After one week of differentiation, the myofibers display contractility, aligned sarcomeres and triads, as well as peripheral nuclei. The entire differentiation process can be followed by live imaging or immunofluorescence. This system combines the advantages of the existing ex vivo and in vitro protocols. The possibility of easy and efficient transfection as well as the ease of access to all differentiation stages broadens the potential applications. Myofibers can subsequently be used not only to address relevant developmental and cell biology questions, but also to reproduce muscle disease phenotypes for clinical applications.

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 ...

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 ...

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

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

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

基于荧光测定活细胞中的钙进入商店经营：从培养的肿瘤细胞，骨骼肌纤维

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

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 ...

Functional Characterization of Endogenously Expressed Human RYR1 Variants

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients&#39; cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.

内源性表达人RYR1变异体的功能表征

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia ...

医学

DNA Electroporation, Isolation and Imaging of Myofibers

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

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

DNA电穿孔，肌纤维的分离和成像

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

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

使用纳米-牛顿分辨率力转换器从骨骼肌肉活检中隔离肌异物并确定收缩函数

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

分离高质量鼠心房和心室肌细胞，同时测量 Ca2+ 瞬变和 L 型钙电流

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

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 ...