作者要感谢Demêmes Danielle和道格拉斯Cotanche和他们的努力，在教学中我们Corti器的隔离方法。此外，我们想感谢electroporator电极以实玛利STEFANOV瓦格纳工程;雪利酒林为她作出贡献的艺术品视频动画和马修长安，贾森米克，和肯德拉马歇尔（ <a href="http://www.goodfightproductions.com">www.goodfightproductions.com</a> ）生产的视频。这项工作是由补助（R03DC010065 - 帕克; P30DC05209 - MEEI听力研究的核心支持; RO1DC007174边）从NIDCD。

第1天。消毒及镀膜玻璃盖玻片。 <ol><li>在高压灭菌器消毒的玻璃显微镜盖玻片干。 </li><li>放入预先消毒四个孔细胞培养皿中的两个井，灭菌的盖玻片。 </li><li>外套1:1 聚- L -鸟氨酸和层粘连蛋白的盖玻片辅以20％ 小牛血清（FBS），过夜4 ° C。 <ol class="lalpha"><li> 400μL的聚- L -鸟氨酸（0.01％溶液保存在4 ° C） </li><li> 400μL，层粘连蛋白（50μg/ mL的原液，分装保存在-20 ° C） </li><li> 200μL的FBS（分装储存在-20 ° C） </li></ol></li><li>热消毒清扫工具，在150℃培养箱过夜。 </li><li>使培养基中含10％血清和10毫克/ mL氨苄青霉素。 <ol class="lalpha"><li> 90毫升贝科的改良Eagle培养基 </li><li> 5毫升FBS（分装储存在-20 ° C） </li><li> 5毫升马血清 （分装储存在-20 ° C） </li><li> 10μL， 氨苄青霉素 （10毫克/毫升原液保存在4 ° C） </li></ol></li></ol>第2天。 Corti器的分离。 <ol><li>消毒正流夹层罩 。 <ol class="lalpha"><li>紫外线灯开启20分钟</li><li>喷雾用70％乙醇的所有表面和使用前，等待5分钟。 </li></ol></li><li>杀头鼠标小狗（P4），在使用操作剪刀孔万能的基础。 </li><li>简单冲洗10厘米，含70％乙醇的菜头。 </li><li>使用手术刀刀片取出表皮。 </li><li>使用手术刀刀片沿矢状缝合打开颅骨，然后平分前脑。保留进一步剥离尾鳍前脑。 </li><li>删除使用钝性分离的前脑，小脑和脑干。 </li><li>取出颞骨（图2a），浸在70％的乙醇短暂，他们转移到无菌的HBSS 3毫米菜含有。 </li><li>使用镊子 ，取出泡从颞骨的岩部分及周围组织。 </li><li>找到的海螺形的耳蜗（图2b）和前庭系统使用镊子分开。 </li><li>在这一发展阶段，骨迷路是没有完全钙化和很容易使用镊子解剖。开始在皮基底和顶部使用镊子仔细分离取出耳蜗骨迷路。 </li><li>螺旋韧带及附着器官尔蒂是沿modiolus（图2C）螺旋盘绕。小心取出，通过确保在使用产钳的基础钩地区螺旋韧带和平仓，当您移动顶部Corti器。 </li><li>在该基地开始，删除螺旋韧带的尔蒂用细镊子＃55（图2D）的器官。 微型隔离尔蒂感觉上皮（可选）的器官 。 </li><li>删除尔蒂钩地区使用两个基地的器官半永久连接的U - 100 28G ½银针产钳CC胰岛素注射器。 </li><li>在心尖开始，请从排内毛细胞，螺旋缘和基部进行（图2E - F）。 电镀尔蒂外植体的器官</li><li>从文化井取出poly-L-ornithine/laminin/FBS的解决方案和培养液中添加130μL。 </li><li>尔蒂解剖器官转移到镀膜玻璃盖玻片和东方文化的外植体，使毛细胞的纤毛向上。 </li><li>取出培养液中，用200微升移液器。确保基底膜使镀膜玻璃盖玻片固体接触。 </li><li>小心加130μL培养基Corti器，使用200毫升吸管。应用到两滴Corti器表面，然后慢慢加入剩余量的盖玻片一侧。确保Corti器，不浮在文化传媒，但仍贴盖玻片。 </li><li>孵育过夜，在37 ° C在5％的CO 2的存在。 </li></ol>第3天。记者到尔蒂培养器官的基因的电。 <ol><li>删除尔蒂文化机关的培养基。 </li><li>加入130μL的H 2 O为1分钟，然后取出使用200微升吸管。 </li><li>加入30μLDsRed的报告质粒（2毫克/毫升H 2 O储存在-20 ° C）。 </li><li>提前electroporator使用显微电极，使阳极一个ND阴极文化两侧。 </li><li>生成一个脉冲（27V，30毫秒，10个脉冲串）electroporate尔蒂植文化机关的记者基因。 <ol class="lalpha"><li>可选：扭转蜗轴和螺旋韧带两侧的外植体，以确保转基因电脉冲的极性。 </li></ol></li><li>等待5分钟。 </li><li>加入130μL的Fugene 6：DNA溶液 （3个部分Fugene 2部分的DNA）。这个解决方案应该是电过程的开始（20步）3 mL 的圆底聚苯乙烯试管中使用层流罩前的准备。为了使这个解决方案： <ol class="lalpha"><li>添加2.4μLOPTI - MEM（存储在4 ° C）的试管。 </li><li> Fugene 6试剂0.6μL（保存在4 ° C）加入到试管中。确保直接添加的Fugene OPTI - MEM，避免直接接触试管双方。 </li><li>涡1秒。 </li><li>在室温下孵育5分钟。 </li><li>添加2.0μL，DsRed的记者质粒双链DNA（储存在-20 ° C，在100微克DNA / mL的H 2 O分装） 。 </li><li>涡1秒。 </li><li>在室温下孵育15分钟。 </li><li>加入200μL培养基。 </li><li>涡1秒。 </li></ol></li><li>在37℃，5％CO2孵育过夜。 </li><li>加入2毫升培养基以及文化和孵化为37 ° C，时间为10天。 </li></ol>代表性的成果我们目前的一个孤立的器官尔蒂从围产期鼠标的方法。 ，在这一点骨迷路变得足够钙化呈现剥离繁琐的过程，可用于小鼠胚胎一天作为年轻16日龄约6。一旦解剖Corti器，它可镀和培养的全部内容（图3）或微孤立的感觉上皮（图4）。我们进一步提出了一种技术来表达外源基因在培养尔蒂机关。许多其他类型的研究，如分析尔蒂基因的表达，采用RT - PCR或原位杂交，共培养尔蒂器官螺旋神经节细胞或外源性干细胞使用的微型隔离器官器官的文化是非常有用， 3，或在体外毛细胞死亡和再生的分析。 <a href="https://www.jove.com/files/ftp_upload/1685/1685fig1.jpg"><img src="/files/ftp_upload/1685/1685fig1tmb.jpg" alt="图1" border="0" /></a> 图1。横截面的P4尔蒂小鼠器官。 （一）从一个cryosectioned耳蜗底回的一个截面从一个P4鼠标获得说明了在本议定书中所描述的小鼠耳蜗一般结构。 Scala的媒体是由螺旋韧带和血管纹横向接壤，Reissner变分的膜优越，螺旋缘内侧，和下方的基底膜。框表示在B（二）扩大该地区的Corti器位于优越​​的基底膜上，并包含一个行内毛细胞，三排的外毛细胞，和各自的支持细胞。虚线1表示尔蒂清扫机关在此期间拆除沿基底膜的位置。虚线2表示沿基底膜是在微分离过程中删除的位置。绿色表示calbindin免疫标签，标签螺旋缘齿间细胞，耳蜗毛细胞，螺旋神经节神经元，以及细胞螺旋韧带和血管纹7。 DAPI核标签为蓝色。 <a href="https://www.jove.com/files/ftp_upload/1685/1685fig2.jpg"><img src="/files/ftp_upload/1685/1685fig2tmb.jpg" alt="图2" border="0" /></a> 图2。从影随行尔蒂清扫突出的器官器官。尔蒂清扫图片）耳蜗及前庭系统位于颞骨内孤立的（红色），B）耳蜗骨迷路，C），螺旋韧带和连接切除器官后，去除的骨迷路，D）的尔蒂螺旋韧带和血管纹（红色），从器官的尔蒂，E）微隔离的感​​觉上皮螺旋缘（红色），和F）隔离螺旋缘（左）和感觉上皮（右）。 <a href="https://www.jove.com/files/ftp_upload/1685/1685fig3.jpg"><img src="/files/ftp_upload/1685/1685fig3tmb.jpg" alt="图3" border="0" /></a> 图3。尔蒂外植体培养的器官 。DIC的图像显示的器官尔蒂的一个P4 Atoh1的nGFP鼠标被描述为五个天的隔离，镀金，并培养。这款鼠标具有经过基因工程改造，使细胞表达亲毛细胞无调性同源基因1（又名Atoh1的Math1）表现出绿色荧光蛋白是局部性核8。尔蒂机关从这些小鼠表现出核GFP标签的所有头发细胞的细胞核中，因此允许为容易使用啶显微镜的感觉上皮的可视化。可以看到比较大的螺旋缘外侧的感觉上皮。 Corti器起源的间质细胞迁移距​​离外植体。蓝核标签的DAPI。 <a href="https://www.jove.com/files/ftp_upload/1685/1685fig4.jpg"><img src="/files/ftp_upload/1685/1685fig4tmb.jpg" alt="图4" border="0" /></a> 图4。微孤立的感觉上皮 。啶形象从感官上孤立的描述和培养过夜，已经从一个P4尔蒂小鼠器官的上皮得到的。微隔离，然后固定在4％多聚甲醛和肌球蛋白7A标签耳蜗毛细胞免疫标记处理。注意从图3的情况下较大的螺旋缘。蓝核标签的DAPI。 <a href="https://www.jove.com/files/ftp_upload/1685/1685fig5.jpg"><img src="/files/ftp_upload/1685/1685fig5tmb.jpg" alt="图5" border="0" /></a> 图5。 DsRed的记者到尔蒂培养器官基因的电。尔蒂整个器官，从P4 Atoh1的nGFP鼠标幼仔被隔离，镀金，然后描述，然后一个啶显微镜下看DsRed的记者基因电穿孔。尔蒂植体器官的细胞表达的转基因DsRed的记者蛋白表现出的感觉上皮的展示一个绿色的核荧光红色荧光和内源性细胞。转基因细胞可以看出，整个螺旋缘和感觉上皮。

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有几个细节，此过程成功的关键。从颞骨隔离尔蒂孵化机关，机关将附加到盖玻片和可行的机关文化的机会就越大时间越短。因此，重要的是要限制清扫术，放置在孵化器的机关之间的时间量。抗生素的选择也是至关重要的，因为许多氨基糖甙类抗生素耳毒性，并会导致毛细胞死亡。虽然这是最好完全放弃使用抗生素，这使得公开污染的可能性。因此，我们建议使用10μg/ mL氨苄青霉素作为一般规则，以克服潜在的污染问题。 这个最麻烦的方面，和其他Corti器的主要文化程序，是机关的倾向浮板孵化过程中。虽然浮动机关文化可以保持5-7天可行的，也有浮动机关培养的弊端。举例来说，浮动机关文化往往把自己折后4-5天，使显微镜问题。随后，器官的结构完整性，可以成为损害时相比，已贴盖玻片机关。我们发现，下面的技术帮助，以确保Corti器不浮在文化传媒，但仍贴盖玻片。首先，大衣的玻璃盖玻片，在1:1 polyornithine /层粘连蛋白补充与所述20％胎牛血清。孵育过夜，在本议定书中所要求的是最短的时间，应涂板。在我们的实验室中，我们经常涂层板，我们需要一个星期，和他们保持在4 ° C直到前一天，当我们使用移动孵育过夜，他们的孵化器。其次，机关后，被运送到涂盖玻片，东方外植体，使毛细胞纤毛面对。这种倾向将有利于坚持基底膜培养皿。第三，取出内以及媒体贴上外植体的涂层盘。这将确保培养皿和基底膜之间的接触和加强外植体的能力，坚持以玻璃。这也将有助于在保持毛细胞行结构的完整性。最后，小心滴到尔蒂使用200μL容量移液器器官表面的培养液2滴，然后慢慢填井，滴在盖玻片一侧的剩余量（共130μL）。重要的是要迅速开展工作，以确保Corti器附件涂盖玻片。从启动培养外植体解剖，通常需要10分钟为一个实行经营者来完成这个尔蒂分离过程的器官。 在这个协议中，我们还提供了一个螺旋缘，Corti器感觉上皮的微型隔离的方法。在此过程中，螺旋缘解剖远离使用28G的感觉上皮半胰岛素针清扫工具。导致微隔离由毛细胞和其相应的支持细胞（图3）行。孤立的感觉上皮然后可以培养，在本议定书中所述。这种微型隔离程序应该比较的感觉上皮细胞从周围组织4的酶分离。在哺乳动物，以及非哺乳动物物种，如鸡，结果从地下室4间质细胞在感觉上皮隔离孤立的前庭器官的嗜热消化。在大鼠耳蜗，在更大的上皮嵴的分离，小上皮嵴和陪同感觉上皮基底膜5嗜热消化的结果。不过，目前还不清楚是否耳蜗感觉上皮可以连接到没有随行的间质细胞的涂层钢板。虽然机械的微型隔离法和酶消化法在螺旋缘的感觉上皮细胞的分离方法的结果，在嗜热消化的微型隔离清扫的优势包括一个相对较短的协议，更便宜的试剂，和潜在的少压力由于消化酶的影响外植体。此外，基底膜是原封不动这种方法，可提高感觉上皮的培养板中的附件。这种方法的缺点包括需要发展的技能，这种微妙的解剖和潜在的机械性损伤的感觉上皮，导致从微观剥离。 作为此过程中实用的例子，我们也提出一个例子使用的器官尔蒂文化;电外源基因进入植文化。上文所述的电程序是基于以前的方法尔蒂电机关。值得注意的是，郑和高（2000）描述尔蒂大鼠离体器官植举行为电的琼脂糖凝胶型槽，然后对胶原蛋白的镀涂层8以及LabTek在无血清培养基2中的幻灯片的电。他们的方法的优点是面向机关，使外植体的顶部表面面对阴极，理论上应导致均匀分布在整个外植体电穿孔细胞。然而在我们手中，我们所描述的方法改善，在此过程中，因为整个过程中Corti器仍然贴盖玻片，从而降低了电后的外植体的操纵。此外，尔蒂机关中所占比例仍较高盖玻片，使用这种方法的。我们的方法是采取从琼斯等人 （2006年），其中使用Fugene 6试剂除了增加了电后基因表达的效率。琼斯等人 （2006年）的协议，机关电穿孔，孵育5分钟，100μLFugene 6转染试剂，镀6。我们的方法不同，在使用3:2的比例，我们发现，经验，提供最低的器官毒性最佳的转基因表达质粒DNA的Fugene 6试剂。我们不使用未经稀释的Fugene 6试剂，这可能会导致中毒性的文化。在我们的协议的电极配置，以及Jones 等人（2006年），在主基因的表达在螺旋缘或感觉上皮取决于阴极的位置结果。虽然有阴极遥远的文化侧面DsRed的阳性细胞，有较高的转基因细胞的浓度接近阴极。要确保完成尔蒂外植体的器官，双方的转基因表达，可以扭转目前的第二个脉冲列车通过简单的反相的线索。在整个尔蒂（ 图 5）器官的转基因表达强劲的协议结果。

<table border="1">
	<thead>
		<tr>
			<th>Material Name</th>
			<th>Type</th>
			<th>Company</th>
			<th>Catalogue Number</th>
			<th>Comment</th>
		</tr>
	</thead>
	<tbody>
		<tr>
			<td>Glass microscope coverslips</td>
			<td>&nbsp;</td>
			<td>DYNALAB Corporation (Rochester, NY)</td>
			<td>2010</td>
			<td>10mm diameter, circle #1, 1mm thickness, 1 ounce</td>
		</tr>
		<tr>
			<td>4 ringed cell culture dish</td>
			<td>&nbsp;</td>
			<td>Greiner Bio-One (Frickenhausen, Germany)</td>
			<td>627170</td>
			<td>Sterilized 35 X 10 mm cell culture dish with 4 inner rings</td>
		</tr>
		<tr>
			<td>Poly-L-ornithine</td>
			<td>&nbsp;</td>
			<td>Sigma-Aldrich Company (St. Louis, MO)</td>
			<td>P4957</td>
			<td>0.01% Solution</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>&nbsp;</td>
			<td>BD Biosciences (Franklin Lakes, NJ) </td>
			<td>354232</td>
			<td>made in mouse</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>&nbsp;</td>
			<td>Invitrogen (Carlsbad, CA)</td>
			<td>26140-095</td>
			<td>Qualified</td>
		</tr>
		<tr>
			<td>Operating scissors</td>
			<td>&nbsp;</td>
			<td>Roboz Surgical Instrument Co. (Gaithersburg, MD)</td>
			<td>RS-6806</td>
			<td>Straight, sharp-blunt length 5"</td>
		</tr>
		<tr>
			<td>#11 Scalpel Blade</td>
			<td>&nbsp;</td>
			<td>Becton Dickenson (Franklin Lakes, NJ)</td>
			<td>372611</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>#4 Dumoxel forceps</td>
			<td>&nbsp;</td>
			<td>Fine Science Tools (Foster City, CA)</td>
			<td>11241-30</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>#55 Dumostar fine forceps</td>
			<td>&nbsp;</td>
			<td>Fine Science Tools (Foster City, CA)</td>
			<td>11295-51</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Modified Eagle Medium </td>
			<td>&nbsp;</td>
			<td>Invitrogen (Carlsbad, CA)</td>
			<td>10564-011</td>
			<td>High Glucose</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>&nbsp;</td>
			<td>Invitrogen (Carlsbad, CA)</td>
			<td>2605088</td>
			<td>Heat Inactivated</td>
		</tr>
		<tr>
			<td>Ampicillin Sodium Salt </td>
			<td>&nbsp;</td>
			<td>Invitrogen (Carlsbad, CA)</td>
			<td>11593-027</td>
			<td>Irradiated</td>
		</tr>
		<tr>
			<td>&frac12; cc Lo-Dose Insulin Syringe </td>
			<td>&nbsp;</td>
			<td>Becton Dickenson (Franklin Lakes, NJ)</td>
			<td>329465</td>
			<td>U-100 28G&frac12;</td>
		</tr>
		<tr>
			<td>Fugene 6 Transfection Reagent</td>
			<td>&nbsp;</td>
			<td>Roche (Mannheim, Germany)</td>
			<td>11-815-091-001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polystyrene test tube</td>
			<td>&nbsp;</td>
			<td>Fisher Scientific (Pittsburgh, PA)</td>
			<td>14-956-5A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>&nbsp;</td>
			<td>The Baker Company (Stanford, ME)</td>
			<td>Model SG603a</td>
			<td>SterileGARD III 
				Advanced Class II 
				Biological Safety Cabinet</td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced-Serum Medium</td>
			<td>&nbsp;</td>
			<td>Invitrogen (Carlsbad, CA)</td>
			<td>31985</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Reporter plasmid</td>
			<td>&nbsp;</td>
			<td>Clontech (Mountain View, CA)</td>
			<td>632539</td>
			<td>pCMV DsRed-Express 2</td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>&nbsp;</td>
			<td>BioRad (Hercules, CA)</td>
			<td>165&#x2013;2662</td>
			<td>BioRad Gene Pulser Xcell</td>
		</tr>
	</tbody>
</table>

primary culture and plasmid electroporation of the murine organ of corti. 

在所有哺乳动物中，试演的感觉上皮位于沿尔蒂螺旋式上升机关内的海螺形的耳蜗的内耳（图1）。在发展中的耳蜗，这是听觉系统的mechanosensory细胞，毛细胞排列在一行内毛细胞和三个基地和中期轮流在心尖转外毛细胞行跨度Corti器的长度。大脑可以解释成神经冲动，基底膜毛细胞转导的声音引起的机械振动。大多数病例是感音神经性聋的耳蜗毛细胞的死亡或功能障碍引起的。 在听觉的研究越来越重要的工具是隔离， 并在1,2,9的器官外植体的体外培养。一旦隔离，外植体可利用在几个方面提供有关规范，异常，或治疗生理。基因表达，纤毛蠕动，细胞和分子生物学，以及毛细胞再生的生物方法尔蒂植器官的实验应用的例子。 这个协议描述尔蒂从新生小鼠的器官的孤立和文化的一种方法。影随行，包括逐步指示，从老鼠幼仔的颞骨的孤立，和随后的耳蜗螺旋韧带，Corti器隔离。一旦隔离，可镀感觉上皮，并在整个体外培养，或作为进一步解剖微隔离，缺乏螺旋缘和螺旋神经节神经元。使用这种方法，小学外植体可维持7-10天。作为此过程中实用的例子，尔蒂外植体的器官会被电外生DsRed的记者基因。这种方法提供了一种比其他方法的改进，因为它提供了隔离，显微切割和Corti器的主要文化，重现性好，毫不含糊，并逐步的方向。

Watch this Scientific Journal Video about Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. at JoVE.com

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

此过程描述了一个隔离带或不带螺旋缘和螺旋神经节神经元的小鼠器官的尔蒂文化的方法。我们也展示了电尔蒂植器官的外源性记者基因的表达方法。

原代培养及质粒电穿孔尔蒂小鼠器官。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

primary-culture-plasmid-electroporation-murine-organ

Research

JoVE Journal

Biology

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

25.7K 次观看.  Harvard Medical School. 此过程描述了一个隔离带或不带螺旋缘和螺旋神经节神经元的小鼠器官的尔蒂文化的方法。我们也展示了电尔蒂植器官的外源性记者基因的表达方法。

视频: Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

电穿孔分枝杆菌

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

科研

生物学

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 ...

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

成年大鼠心脏心肌细胞的原代培养

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

大鼠肝细胞的分离和小学文化

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

小鼠原代胰腺腺泡细胞的分离和培养

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder diseases.

电穿孔对小鼠膀胱 Urothelium 质粒 DNA 传递的新方法

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

从成人小鼠腭中分离和培养原代口腔角质形成细胞

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in mice.

将质粒DNA电穿孔到小鼠骨骼肌中

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological ...

揭开炎症性关节炎的面纱：探索滑膜细胞的功能

Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue

Rheumatoid arthritis is an autoimmune disease that leads to chronic inflammation of joints. Synovial macrophages and synovial fibroblasts have central roles in the pathogenesis of rheumatoid arthritis. It is important to understand the functions of both cell populations to reveal the mechanisms underlying pathological progression and remission in inflammatory arthritis. In general, in vitro experimental conditions should mimic the in vivo environment as much as possible. Primary tissue-derived cells have been used in experiments characterizing synovial fibroblasts in arthritis. In contrast, in experiments investigating the biological functions of macrophages in inflammatory arthritis, cell lines, bone marrow-derived macrophages, and blood monocyte-derived macrophages have been used. However, it is unclear whether such macrophages actually reflect the functions of tissue-resident macrophages. To obtain resident macrophages, previous protocols were modified to isolate and expand both primary macrophages and fibroblasts from synovial tissue in an inflammatory arthritis mouse model. These primary synovial cells may be useful for in vitro analysis of inflammatory arthritis.

作者聚焦：从小鼠关节炎组织中分离和培养原代滑膜巨噬细胞和成纤维细胞  

The present study provides a modified protocol to isolate synovial macrophages and fibroblasts from murine inflammatory arthritis tissue.

小鼠关节炎组织中原代滑膜巨噬细胞和成纤维细胞的分离和培养

Rheumatoid arthritis is an autoimmune disease that leads to chronic inflammation of joints. Synovial macrophages and synovial fibroblasts have central ...