作者希望感谢许多扩展到博士。何塞普普拉纳斯和胡安·卡斯蒂略为他们的专业知识，在这种文化协议的开发和应用，以小型鱼类和两栖类动物。还要感谢的无数个人谁不知疲倦地帮助肌肉组织的许多鱼（无论是在种类和数量），包括马修充电，Delci克里斯滕森，扎卡里福勒，布鲁克弗伦岑，弥敦道弗勒利希，基拉马歇尔的解剖和分离，本·迈耶，伊桑Remily和Sinibaldo罗梅罗。这项工作是由阿拉巴马大学生物启动资金的伯明翰部，中心蛋白酶研究美国国立卫生研究院资助＃2P20 RR015566，美国国立卫生研究院NIAMS格兰特＃R03AR055350支持，并NDSU前进NSF资助＃HRD-0811239到PRB。技术支持也由UAB的营养肥胖研究中心奖＃P30DK056336，美国国立卫生研究院NIDDK提供， 其内容完全是作者的责任，并不一定代表了美国国立卫生研究院的官方意见。

伦理声明:所有涉及本文所述脊椎动物实验是事先由阿拉巴马大学伯明翰分校的机构动物护理和使用委员会，并与实验动物福利，健康美国全国学院办公室既定准则卫生与人类服务部。 1，文化准备<ol><li>制备基础培养基如下:9毫米的NaHCO 3（每2升1.51克），20mM的HEPES（每2升9.53克）的DMEM（13.48克/ L，每2升26.96克）。 <ol><li>确定pH值调整到7.40，用HCl或NaOH。 </li><li>确定重量克分子渗透压浓度，并调整至约300毫渗透摩尔浓度用NaCl（如果需要）。 </li><li>过滤消毒使用（2）避光长达2-3个月的1升真空灭菌系统和储存在4℃。 注:请参阅表1和表2为完整的试剂，工具，consum埃布尔斯和设备的信息。 </li></ol></li><li>通过在50ml的超纯无菌水溶解5毫克γ-照射的聚-L-赖氨酸（&gt; 300,000 MW）制备多聚-L-赖氨酸处理过的平板上。轻轻混匀颠倒在室温下10分钟，确保赖氨酸聚合物充分溶解。 </li><li>吸取的聚-L-赖氨酸溶液中的所需量在每孔中，根据盘的类型（ 表3）。让板站在层流罩5分钟。 </li><li>接着，除去聚-L-赖氨酸溶液中，用无菌水洗涤两次。让板在空气中干燥在层流罩20分钟。 </li><li>得到1毫克层粘连蛋白（Engelbreth-的Holm-群原点）和在50ml基本培养基中溶解至20微克/毫升的最终浓度。应用层粘连蛋白溶液，以聚-L-赖氨酸处理过的细胞培养板中以2微克/厘米2。 （请参阅表3为不同印版尺寸） </li><li>轻轻摇动的解决方案，以确保福LL很好的覆盖。密封板与实验室的磁带并将其放置在培养箱中培养24小时。孵化器应设置适当的温度下被使用的物种（ 见表7），并且没有额外的气体需要被添加到孵化器。 注:层粘连蛋白，必须允许坚持以板为种子细胞前24小时。如果不这样做会导致较差的无细胞的黏附。 </li><li>如表4所示准备媒体。完整的媒体使用的当天最好的准备。 </li><li>在筹备组织分离，收集下列项目和高压灭菌:300-500毫升烧杯（次）;玻璃培养皿;手术刀处理;镊子（粗，细）;解剖剪刀;和几张斥水高压釜纸。 注:对于每个人协助解剖过程中，（2）手术刀把手，（1）镊子（类型根据个人喜好），（1）解剖剪刀，和斥水高压灭菌纸（5-10）表是足够了。 </li><li>除了以上的高压蒸汽灭菌的物品，组装70％乙醇的重量平衡，无菌的50ml锥形试管（数量取决于培养的大小），和冰。 </li></ol> 2，组织解剖<ol><li>在开始之前，请确保鱼了充足的库存可用。 注:要使用的鱼的年龄是至关重要的。而两种不同种类的鱼可能是类似的权重，一个物种一个年轻的鱼将拥有比其他种类的旧鱼更多的MPC。作为一般规则，年轻的鱼，用鲑工作时，特别是最好的。对于danionins，鱼一样古老，按一年可以得到最佳利用，而年龄4-6个月或更年轻（最多15克）鲑鱼是最优的。 </li><li>每次5克组织被解剖，等分25毫升隔离介质的进入无菌50ml锥形管中。 </li><li>称重，记录质量和数量管（S）。放置管置于冰上。 </li><li>安乐死2-3鱼在同一时间通过浸渍在&gt; 300毫克/升碳酸氢钠缓冲三卡因甲磺酸盐。允许鳃盖运动停止时间&gt; 5分钟，然后在70％的乙醇（包含在无菌烧杯中），持续30秒浸没鱼。 </li><li>从70％的乙醇除去鱼，并放置于斥水高压釜纸。直接背后的鳃盖，用手术刀做一个肤浅的，浅薄的切口。抓住皮肤在上述切口去除鳞屑和皮肤拉扯，鱼的尾巴。 </li><li>下面的皮肤切除，切除epaxial，鱼从两侧快速糖酵解&#39;白&#39;肌肉（避免缓慢氧化"红色"肌肉位于侧线附近），并将其放置在分离培养基。所要求的机构丢弃鱼的其余部分。 注:虽然这是完全有可能的文化多用途储值卡从红肌原，几乎每一个使用硬骨鱼类的MPC出版以来，利用细胞从epaxial米隔离yotome。 </li><li>直到获得肌肉足量，重复步骤2.4-2.6。在清扫，确保汇集肌肉组织仍然在冰上以保持细胞活力。 </li></ol> 3，机械解离<ol><li>倒入25毫升/5克肌肉组织中的一个管到玻璃培养皿。使用两个手术刀手柄附带＃10手术刀片，肉末组织的浆液或菜泥的一致性。拉动手术刀过去对方一个"背的往复"的运动效果最好。 注意:虽然组织匀浆可能看起来合适，活细胞的数目将被显着降低，如果不废除。 </li><li>用25毫升血清吸管和血清学移液器，取出组织成浆，并替换原来的锥形管。 </li><li>重复步骤3.1-3.2，直到所有的管子都组织已被机械分离。 </li><li>离心机组织在300×g离心5分钟，10°C。 </li><li>傅杰仕由于离心，弃去25毫升培养基上清液用25毫升血清吸管，真空吸引器，或通过仔细调迁。 </li><li>加25毫升洗涤介质的每个管，重悬组织，并如上述recentrifuge。 </li><li>用洗涤介质洗涤两次，每次除去上清液。 </li></ol> 4，酶解<ol><li>在步骤3.4中，通过合并将0.22g IV型胶原酶和44毫升基础培养基中（或足够数量的0.11 g/0.22毫升）准备胶原酶溶液。 </li><li>在4℃下搅拌的烧杯中10-15分钟过滤消毒用50毫升的注射器和0.45μm的注射器过滤器。 注意:一个以上的注射器过滤器可根据需要，根据不同的胶原酶制备。从厂商比沃辛顿生化等获得胶原酶造成过滤时更加困难。胶原酶用于分离肽键在构成肌内膜胶原蛋白。这消化将移除肌纤维的保护屏障。 </li><li>对于每个克肌肉组织，在0.2％胶原酶溶液重新悬浮组织。对于5克肌肉组织，加入10毫升胶原酶溶液和15毫升的基础培养基。 </li><li>加入10微升的PSF每毫升胶原酶溶液/基础培养基（ 例如 250微升至25毫升）。确保组织很好暂停，因为这会影响酶消化的效率。 </li><li>孵育肌肉组织中胶原酶60分钟，在18℃，温和的摇摆。取决于机械解离和物种的程度，胶原酶消化可以延长至90分钟，尽管这可能会影响细胞活力。 </li><li>下面胶原酶消化，离心锥形管，在300×g离心5分钟，在12°C。丢弃在25ml洗涤介质的上清，重悬组织。 </li><li> Recentrifuge在300×g离心5分钟，在126，C。用洗涤介质重复第二次。 </li><li>洗涤后的组织两次，重悬肌肉组织在25ml洗涤介质和磨碎的与10ml的血清吸液管，直到组织/培养基中匀浆通过在吸液管和出相对容易。 5-10 triturations通常是足够的。 <ol><li>重复用5毫升的血清吸液管，然后连接到一个注射器的16号金属套管。 </li></ol></li><li>离心机锥形管在300×g离心5分钟，在12°C。弃去上清液。 </li><li>在上述5分钟离心，结合0.25克胰蛋白酶用5毫升基础培养基的制备胰酶消化解决方案。 </li><li>搅拌，在4℃下进行10-15分钟，并过滤消毒用20毫升注射器和0.45微米注射器过滤器。 </li><li>检索锥形管中并加入100μl的胰蛋白酶溶液和4.9毫升解离介质的每1克肌肉组织的每管中。对于EXA投影机的简易，加入500μl的胰蛋白酶溶液和24.5毫升分离培养基5克肌肉组织的。 注:胰蛋白酶是一种丝氨酸蛋白酶，将分离生肌前体细胞从基底层。 </li><li>在胰蛋白酶/解离重悬组织培养基好。孵育20分钟，在18°C。 </li><li>继第一胰蛋白酶孵育，离心锥形管，在300×g离心1分钟，在12°C。 </li><li>通过组合与分离培养基的上清液在1体积的上清液，以4倍体积的隔离介质的浓度中和胰蛋白酶。在4°C存储第二胰蛋白酶消化过程中。 </li><li>到组织的剩余粒料，重复步骤4.12-4.15，合并上清液以下步骤4.15从第一胰蛋白酶消化上清液/隔离介质。 </li><li> 或者:胶原酶和胰蛋白酶消化可以被组合以最大化电池的产量。 &lt;醇&gt; <li>要做到这一点，磨碎的胶原酶消化使用连接到20ml的注射器的金属套管（6在长度和16号的效果最好），直至匀浆轻易地穿过套管。 </li><li>到匀浆，加入500μl的胰蛋白酶溶液（步骤准备4.10）和孵化在18℃下20分钟。 </li><li>在孵育，离心锥形管，在300×g离心1分钟，在12°C。 </li><li>通过组合与分离培养基的上清液在1体积的上清液，以4倍体积的隔离介质的浓度中和胰蛋白酶。 </li><li>在4°C存储第二胰蛋白酶消化过程中。 </li><li>继续执行步骤4.18与标准协议。 </li></ol></li><li>免除了最终的上清液/分离培养基混合物倒入50ml锥形管和离心300×g离心 对于在12°C。10分钟</li><li>删除注意不要扰乱细胞上清液球团矿。移液器2毫升完全培养基到每个管中，溶解各细胞沉淀。 </li><li>结合的细胞悬浮于完全培养基中1 50ml锥形管中。 </li><li>冲洗每管1 ml完全培养基，用细胞悬浮以前的池相结合。 </li><li>磨碎与金属套管和注射器的5-10倍。 </li><li>通过100微米的细胞滤网过滤细胞悬液，用完全培养基冲洗。 </li><li>通过一个40微米的细胞过滤筛选的细胞悬液。 </li><li>加足够的完全培养基至50ml离心和在300×g离心10分钟，在15℃下</li></ol> 5，计数，稀释和细胞种植<ol><li>下面的步骤4.25最后离心，通过仔细的调迁或用血清学移液器取出上清液。重悬细胞沉淀在5ml完全培养基。 </li><li>随着细胞充分悬浮，去除20微升将细胞悬浮液并置于1.5ml的微量离心管中; </li><li>加入5微升台盼蓝染色，并等待5分钟。使用血细胞计数器，测定存活细胞的数量。 </li><li>一旦确定，稀释细胞至所需的浓度。硬骨鱼类的MPC最好是在每平方厘米2 150,000-200,000细胞接种。对于6孔镀策略，稀释细胞至1.5×10 6 -每毫升2.0×10 6支持足够的增殖和分化。 </li><li>检索聚-L-赖氨酸处理过的，层粘连蛋白包被的板和种子细胞。 （ 见表5板专用稀释液和电镀卷。）注: 表6描述了细胞每克肌肉组织从不同的硬骨鱼类物种中分离的孤立的平均数。虹鳟鱼（O. mykiss）的表现出每克肌肉组织较少的多用途储值卡做比斑马鱼（ 斑马鱼 ），并将于从而需要更多的鱼进行适当的播种隔离。 </li><li>密封板与实验室的磁带并将其放置在冷藏保温箱在适当的温度。此处列出（除小鼠）所有物种的MPC可能没有二氧化碳（CO 2）补充正常大气条件下进行培养。 （ 见表7）。 </li><li> 或者:一些实验室都采用了协议，要求允许多用途储值卡坚持以培养基质40分钟。 <ol><li>然后冲洗板用洗涤介质以除去任何松散地附着和非贴壁细胞。注意:此步骤是非常重要的，以避免其他细胞的"非特异性"的附着力（ 如成纤维细胞）的培养基质。 </li><li>添加完整的媒体和地点的细胞在孵化器和照顾，详见下文。 </li></ol></li></ol>

没有什么可以透露。

生肌方案，在任何检查的物种，可以最方便地通过一个体外系统的研究。事实上，在隔离，生肌前体细胞（MPC）中的鱼或myosatellite细胞（MSCs）在哺乳动物中很容易进入这个涉及细胞增殖，细胞周期停药，并成肌细胞的终末分化和融合的成肌成肌管新生的高度管制的过程。普遍缺乏鱼品种的转基因报告基因的菌株（与斑马鱼67和虹鳟鱼69可能是个例外）限制在 MPC / MSC活...

哺乳动物肌肉发生的相当的了解已经通过这个过程的概括在这两个初级小鼠（ 小家鼠 ）的成肌细胞培养物和良好描述的小鼠衍生的细胞系中获得，C2C12 5。在20世纪50年代6起，这些文化都引发了很多进步的murinemyogenic程序的理解，并推而广之，肌肉发生在其他脊椎动物。此外，单细胞的肌纤维植技术已增加了相互作用的了解卫星细胞和周围的肌纤维7-9之间。细胞培养物对肌肉发育的研究特别有吸引力因短时间从前驱分化的细胞10，转染的相对容易的RNAi 11-14，转基因15,16和表达的研究14,17,18， 在体外扩增后回输体内 18-20，甚至排版埃里森生肌前体细胞及其调节剂等不同分类群21,22。而由于培养系统的人工环境的差异进行了说明5,23，这些体外系统已被证明是不可缺少的，以我们的管的多核形成错综复杂的程序的夹层，终末分化的单核myofibersfrom称为myosatellite增殖祖细胞细胞（MSCs）的哺乳动物之一。 类哺乳动物之外，然而，控制肌肉发育机制，保护和/或发散了解甚少，主要是由于来自不同类群培养生肌前体细胞（MPC）中和成肌细胞的难度。事实上，主要的成肌细胞培养只被三鸟24-26一爬行动物27，少数两栖动物28-30，和一些鱼类1,3,4,31-33描述。从已经连续肌细胞系rtebrates比鼠类34-36其它更是少见，唯一的非哺乳动物肌细胞系被来自日本鹌鹑（Cortunix粳稻 ）得出，QM7 37。尽管很多人试图在永生化，一个硬骨肌细胞系仍然是难以捉摸和协议，用于这些细胞的转染效率才公布，今年15。因此，非常需要从多种脊椎动物的培养主要的MPC和成肌细胞清楚和优化的协议，不仅进一步扩大我们的生肌程序的演变知识，而是采用比较生理学的力量取得突破的人骨骼肌疾病和病症的治疗。 虽然文献中的MPC /成肌细胞分离38-49很多报道，这是很常见的作家来描述简单，往往不完整，格式如隔离的协议。此外，最有启发性的协议回购RTED已经开发了用于小鼠50-53，并且其中的一些依赖于抗体的选择54,55或荧光的转基因56,57，使得这些协议不可用或通过肌肉生物学家使用不切实际的最深处非啮齿类动物物种。随着对鱼，两栖动物，爬行动物肌肉发育，详细和全面的协议，与视听的指导，并与远缘物种证明效率描述鲜为人知的，将是最有帮助的领域。 首先由鲍威尔和他的同事在1989年58所述，下列协议最初是从鲑鱼（即，虹鳟， 虹鳟和大西洋鲑， 大西洋鲑 ）和一些较大的鲤科鱼类（ 如金鱼，Carassiusauratusauratus）隔离的MPC和成肌细胞。 2000年，Fauconneau和Paboeuf优化的主要成肌细胞培养虹鳟鱼59，minoroptimizations马去该协议利用的在亚科分支（斑马鱼， 斑马鱼 ，和巨人斑马，Devario aequipinnatus）32，由于可用于斑马鱼工作的许多遗传工具，因此，其近亲属的几个较小的小鱼。硬骨鱼是有吸引力的生物进行研究，由于其不同的发展战略（至少在大多数物种）。大鲑鱼，最喜欢的鱼类，生长不定，随着到期日由不受约束的渐近增长潜力，即使在年老60-62。与斑马鱼，大danionins如巨人斑马63和小胡子斑马显示增长潜力典型的硬骨鱼，使他们直接相拼理解一个理想的平台是否MPC细胞命运的选择对骨骼肌增生与肥大的作用。 同样地，我们已经证明，这个协议可与小鼠和蝾螈使用，具有相对高的细胞产量和VIABility指数。 Urodele蝾螈，如墨西哥蝾螈（Ambystomamexicanum），具备非凡的能力，再生组织，包括整个四肢和尾巴64-66。这一特点使得这些两栖动物有趣的骨骼肌萎缩和老化模型。使用下面描述的协议，类似的方法可以进行，这已在许多鱼种已经完成，为这类研究更广泛的比较方面。因为许多真正比较生物学家理解的，在基本的生物学和生物医学平移最有意义的进步可以当数据被最广泛的光谱范围内进行分析（在此，整个脊椎动物谱系）制成。

<table border="0" cellpadding="0" cellspacing="0" width="743">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. Detailed Reagent Information</td>
		</tr>
		<tr>
			<td height="42" style="height:43px;width:205px;">Reagent</td>
			<td style="width:217px;">Company (Preferred v. Alternate)</td>
			<td style="width:160px;">Catalog Number (Preferred v. Alternate)</td>
			<td style="width:161px;">Quantity per Culture</td>
		</tr>
		<tr>
			<td height="20" style="height:20px;">&#947;-irradiated poly-L-lysine</td>
			<td>Sigma-Aldrich (MP Biomedicals)</td>
			<td>P5899 (ICN19454405)</td>
			<td>5 mg</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:205px;">DMEM (high glucose)</td>
			<td>Sigma-Aldrich (cellgro)</td>
			<td>MT-50-003-PB (D7777)</td>
			<td>2 L</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:205px;">Laminin</td>
			<td>BD Biosciences (Sigma-Aldrich)</td>
			<td>CB-40232 (L2020)</td>
			<td>1 mg</td>
		</tr>
		<tr>
			<td height="41" style="height:41px;width:205px;">Sodium Bicarbonate (NaHCO3)</td>
			<td>Fisher Scientific (Sigma-Aldrich)</td>
			<td>BP328-500 (S5761)</td>
			<td>1.51 g</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:205px;">HEPES (C8H18N2O4S)</td>
			<td>Fisher Scientific (Sigma-Aldrich)</td>
			<td>BP310-1 (H6147)</td>
			<td>9.53 g</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:205px;">Antibiotic/Antimycotic</td>
			<td>Thermo Scientific (Sigma-Aldrich)</td>
			<td>SV3007901 (A5955)</td>
			<td>17-20 mL</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:205px;">Gentamicin Sulfate</td>
			<td>Lonza (Sigma-Aldrich)</td>
			<td>BW17-519Z (G1397)</td>
			<td>2-3 mL</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:205px;">Donor Equine Sera</td>
			<td>Thermo Scientific (Sigma-Aldrich)</td>
			<td>SH3007403 (H1270)</td>
			<td>75 mL</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:205px;">Fetal Bovine Sera</td>
			<td>Thermo Scientific (Sigma-Aldrich)</td>
			<td>SH3007103 (F2442)</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:205px;">Collagenase (Type IV)</td>
			<td>Worthington (Sigma-Aldrich)</td>
			<td>LS004189 (C9891)</td>
			<td>0.44 g</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:205px;">Trypsin (from Pancreas)</td>
			<td>MP Biomedicals (Sigma-Aldrich)</td>
			<td>ICN15357125 (T5266)</td>
			<td>1 g</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Consumables, Tools and Equipment</td>
		</tr>
		<tr>
			<td height="42" style="height:43px;width:239px;">Consumable</td>
			<td style="width:204px;">Tools</td>
			<td style="width:199px;">Equipment</td>
			<td></td>
		</tr>
		<tr>
			<td height="28" style="height:28px;">Cell Culture Plates</td>
			<td>Forceps (Coarse)</td>
			<td>Serological Pipettor</td>
			<td></td>
		</tr>
		<tr>
			<td height="28" style="height:28px;width:239px;">Sterile 50 mL Conical Tubes</td>
			<td style="width:204px;">Forceps (Fine)</td>
			<td>pH Meter</td>
			<td></td>
		</tr>
		<tr>
			<td height="28" style="height:28px;width:239px;">Laboratory Tape</td>
			<td style="width:204px;">Scalpel Handles</td>
			<td>Chilling Incubator (Echotherm)</td>
			<td></td>
		</tr>
		<tr>
			<td height="28" style="height:28px;width:239px;">0.2 &#956;m Vacuum Sterilization Systems</td>
			<td style="width:204px;">Scalpel Blades (#10, #11)</td>
			<td>Laminar Flow Hood</td>
			<td></td>
		</tr>
		<tr>
			<td height="28" style="height:28px;width:239px;">Water-repellant Autoclave Paper</td>
			<td style="width:204px;">Surgical Scissors</td>
			<td>Vacuum Manifold</td>
			<td></td>
		</tr>
		<tr>
			<td height="28" style="height:28px;width:239px;">Serological Pipettes</td>
			<td>Glass Petri Dishes</td>
			<td>Microosmolality Meter</td>
			<td></td>
		</tr>
		<tr>
			<td height="28" style="height:28px;width:239px;"></td>
			<td>12-16 G Cannulas with Luer Locks</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 3. Optimized Volumes for Coating Cell Culture Plates</td>
		</tr>
		<tr>
			<td height="24" style="height:24px;width:167px;">Plate Size</td>
			<td style="width:192px;">cm^2 per Well</td>
			<td style="width:132px;">Poly-L-lysine*</td>
			<td style="width:145px;">Laminin**</td>
		</tr>
		<tr>
			<td height="20" style="height:20px;">6 well</td>
			<td>9.5</td>
			<td>1.6 mL</td>
			<td>1</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:167px;">24 well</td>
			<td>1.9</td>
			<td>0.32</td>
			<td>0.2</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:167px;">48 well</td>
			<td>0.95</td>
			<td>0.16</td>
			<td>0.1</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:167px;">96 well</td>
			<td>0.32</td>
			<td>0.06</td>
			<td>0.03</td>
		</tr>
		<tr>
			<td height="40" style="height:40px;width:167px;">*0.1 mg/mL concentration</td>
			<td colspan="2">** 0.020 mg/mL concentration</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 4. Media for Isolation, Dissociation, and Culture</td>
		</tr>
		<tr>
			<td height="24" style="height:24px;width:163px;">Reagent</td>
			<td style="width:155px;">Isolation</td>
			<td style="width:127px;">Wash</td>
			<td style="width:127px;">Dissociation</td>
			<td>Complete</td>
		</tr>
		<tr>
			<td height="20" style="height:20px;">Base Medium</td>
			<td>419.25 mL</td>
			<td>395.40 mL</td>
			<td>297.00 mL</td>
			<td>178.00 mL</td>
		</tr>
		<tr>
			<td height="20" style="height:20px;width:163px;">PSF*</td>
			<td>5.00 mL</td>
			<td>4.00 mL</td>
			<td>3.00 mL</td>
			<td>2.00 mL</td>
		</tr>
		<tr>
			<td height="40" style="height:40px;width:163px;">Gentamicin Sulfate**</td>
			<td>0.75 mL</td>
			<td>0.60 mL</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td height="40" style="height:40px;width:163px;">Donor Equine Serum</td>
			<td>75.00 mL</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td height="40" style="height:40px;width:163px;">Fetal Bovine Serum***</td>
			<td style="width:155px;">-</td>
			<td>-</td>
			<td>-</td>
			<td>20.00 mL</td>
		</tr>
		<tr>
			<td colspan="4" height="19" style="height:19px;width:660px;">* PSF: penicillin/streptomycin/fungizone cocktail (100x); ** 50 mg/mL concentration; *** Characterized</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 5. Recommended Dilutions and Plating Volumes</td>
		</tr>
		<tr>
			<td height="24" style="height:24px;width:75px;">Plate Size</td>
			<td style="width:103px;">cm^2 per Well</td>
			<td style="width:145px;">Dilution</td>
			<td style="width:111px;">Plating Volume</td>
		</tr>
		<tr>
			<td height="20" style="height:20px;">6 well</td>
			<td>9.5</td>
			<td>1.5-2.0x10^6 cells/mL</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td height="20" style="height:20px;width:75px;">24 well</td>
			<td>1.9</td>
			<td>1.5-2.0x10^6 cells/mL</td>
			<td>250 &#956;L</td>
		</tr>
		<tr>
			<td height="20" style="height:20px;width:75px;">48 well</td>
			<td>0.95</td>
			<td>1.5-2.0x10^6 cells/mL</td>
			<td>150 &#956;L</td>
		</tr>
		<tr>
			<td height="20" style="height:20px;width:75px;">96 well</td>
			<td>0.32</td>
			<td>1.5-2.0x10^6 cells/mL</td>
			<td>50-100 &#956;L</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 6. Average number of cells per g tissue</td>
		</tr>
		<tr>
			<td colspan="2" height="24" style="height:24px;width:201px;">Species</td>
			<td colspan="2" style="width:273px;">Average # cells/g tissue</td>
		</tr>
		<tr>
			<td colspan="2" height="20" style="height:20px;">Danio rerio</td>
			<td colspan="2">6,400,000</td>
		</tr>
		<tr>
			<td colspan="2" height="20" style="height:20px;width:201px;">Danio dangila</td>
			<td colspan="2">1,783,000</td>
		</tr>
		<tr>
			<td colspan="2" height="20" style="height:20px;width:201px;">Devario aequipinnatus</td>
			<td colspan="2">1,797,000</td>
		</tr>
		<tr>
			<td colspan="2" height="20" style="height:20px;">Oncorhynchus mykiss</td>
			<td colspan="2">66,800</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 7. Recommended Incubation Temperatures</td>
		</tr>
		<tr>
			<td colspan="2" height="24" style="height:24px;width:201px;">Species</td>
			<td colspan="2" style="width:273px;">Temperature</td>
		</tr>
		<tr>
			<td colspan="2" height="20" style="height:20px;">Danio/ Devario spp.</td>
			<td colspan="2">26 - 28 &#176;C</td>
		</tr>
		<tr>
			<td colspan="2" height="20" style="height:20px;width:201px;">Oncorhynchus/Salmo spp.</td>
			<td colspan="2">10* - 18 &#176;C</td>
		</tr>
		<tr>
			<td colspan="2" height="20" style="height:20px;width:201px;">Ambystoma mexicanum</td>
			<td colspan="2">18&#176;C</td>
		</tr>
		<tr>
			<td colspan="3" height="20" style="height:20px;">* Lower temperatures support lower proliferation rates.</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of primary myogenic precursor cell/myoblast cultures from basal vertebrate lineages

由于固有的困难，并参与研究体内生肌程序时，从骨骼肌的居民的成体干细胞来源的原代培养系统，生肌前体细胞（MPC）中，已证明不可或缺到我们的哺乳动物骨骼肌发育的理解和增长。特别是脊椎动物的基部类群，但是，数据是有限的，描述控制的自我更新，增殖和MPC的分化的分子机制。特别令人感兴趣的是背后基底脊椎动物经受相当大的后期幼骨骼肌肌纤维增生（ 即硬骨鱼）和全再生的附属物损失（ 即 urodele两栖动物）的能力的潜在机制。此外，使用培养的成肌细胞可能有助于再生和生肌程序的总结和它们之间的差异的理解。至为此，我们详细描述了一个强大和高效的协议（和其中的变化），用于隔离和维护的MPC和他们的后代，成肌细胞和未成熟的肌管，在细胞培养作为理解生肌计划的发展平台，与更多的开始基底脊椎动物。凭借斑马鱼（ 斑马鱼 ）的模式生物的状态，我们报告这个协议的鲤科鱼类分支亚科的小鱼应用。在串联，这个协议可以被用来从墨西哥蝾螈（Ambystomamexicanum），甚至啮齿类实验动物隔离多用途储值卡，以实现更广泛的比较方法。该协议是目前广泛使用的几种鱼类物种，包括虹鳟，鲑鱼和鲷鱼1-4学习肌肉发育。

二十四小时后播种，生肌前体细胞（MPC）中应该是可见的附着于层粘连蛋白基质（参见图1a和 1d）。以下播种，细胞（MPC）中采用一个纺锤状的形状，表示该信元类型（ 图1），并MYOD1 +（ 图2）。在斑马品种，多用途储值卡显得更紧凑，更小的双极型工艺比执行从虹鳟和褐种多用途储值卡。然而，在四天的文化，所有鱼品种多用途储值卡检...

Watch this Scientific Journal Video about Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages at JoVE.com

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

在体外培养系统已被证明不可缺少的，以我们的脊椎动物肌肉发育的理解。然而，还有许多尚待了解非哺乳动物骨骼肌肉的发育和成长，尤其是在基础类群。一个高效，健壮的协议用于分离该组织中，肌细胞前体细胞（MPC）中的成体干细胞，并维持它们的自我更新，增殖，并在原代培养物分化的设置允许的保守和发散调节机制的识别整个脊椎动物的谱系。

原发性生肌前体细胞/成肌细胞从基底哺乳动物支系文化准备

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

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Biology

20.2K 次观看.  University of Alabama at Birmingham.  在体外培养系统已被证明不可缺少的，以我们的脊椎动物肌肉发育的理解。然而，还有许多尚待了解非哺乳动物骨骼肌肉的发育和成长，尤其是在基础类群。一个高效，健壮的协议用于分离该组织中，肌细胞前体细胞（MPC）中的成体干细胞，并维持它们的自我更新，增殖，并在原代培养物分化的设置允许的保守和发散调节机制的识别整个脊椎动物的谱?...

视频: Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

从晚期果蝇蛹的大脑原发性神经文化

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

科研

生物学

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

从Midgastrula舞台果蝇胚胎神经文化的制备

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

游离的小鼠皮层神经元文化的制备

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

小学游离中脑多巴胺鼠类新生儿的细胞培养

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

2 dGuo治疗胸腺器官培养的制备

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

制作生肌工程的组织结构

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

制备海兔感觉运动神经元细胞培养

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

建立从小学成人成纤维细胞培养鼠害

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.