这项工作是支持由美国国家癌症研究所（K08CA140946，YC），（5F32CA130372，到PC），（R01CA102774和R01HL055748到PB），国防部（PC094302 YC），GIST的研究舒曼家庭基金（以PC）和斯塔尔癌症协会（PC机，YC，CLS和PB）。我们要感谢帮助cryosectioning和免疫卡蒂娅MANOVA，宁番，MSKCC分子细胞学核心设施和Mesurh Turkekul。

1。解剖小鼠胃肠道<ol><li>安乐死小鼠当地的IUCAC指引</li><li>泡沫塑料表面上的每个引脚的四肢和70％的乙醇冲洗腹部表面。从隔膜到耻骨长正中切口打开腹腔。请削减食管远端，在远端大肠的二次切割，整块取出消化道从胃到肛门，仔细切割用小剪刀在十二指肠和盲肠韧带。放置在培养皿中，用PBS消化道。解剖远离肠系膜使用镊子（图1）。胃，小肠，大肠，在幽门和回盲部切分开。冲洗管腔使用5ml注射器喂食针（年纪较大的老鼠）或钝针（年轻小鼠）用PBS胃肠道的每个部分。 </li><li>打开胃，小肠，大肠肠系膜边境沿线。消化道各部分，切两片整装可视化和cryosection之一。 </li></ol> 2。整个安装的样品制备和免疫<ol><li>消化道各部分的整片放入一个1.5厘米的离心管充满至少1小时，用1.0毫升冰冷的丙酮和修复样本在4 ° C。样品可以在-20 ° C保存1周我们通常存储在丙酮中的样品，并着手准备cyrosections。 </li><li>用PBS清洗样品2倍。将样品在培养皿菜用PBS。在“解剖miscroscope”下，小心地刮去用手术刀的镊子一端，留下肌层，粘膜层。 </li><li>肌肉可以切成不同的抗体染色5毫米件。不要存放超过2天的PBS染色前。 </li><li>可以做到在24孔板或在1.5毫升离心管座和孵化步骤。座与封闭液0.5毫升（5％山羊血清，0.1％Triton X - 100的PBS为1小时4 °的彗星）。 </li><li>在封闭液稀释的一抗孵育，和旋转4℃过夜° C。我们已经成功地使用鼠抗套件ACK - 2，兔抗Ano1，和兔抗PGP9.5整装免疫抗体。 </li><li> 5分钟2X用PBS洗涤，在室温条件下肩。 </li><li>孵育二抗封闭液稀释后在室温下2小时上肩。我们通常使用的Alexa Fluor 488抗兔的Alexa Fluor 594抗鼠抗体。 </li><li> 3X 15分钟，用PBS洗涤，在室温条件下肩。 </li><li>幻灯片上，顶部浆膜侧山镜，以确保在组织的方向。这一方针将确保浆膜侧面对盖玻片和遇到的第一个，作为一个载玻片重点。我们在解剖显微镜。有时候，我们简要分析荧光显微镜下，以正确东方的组织。在荧光显微镜可视化工具包或Ano1下，应该首先从盖玻片一侧遇到的ICC我的网络。接下来，吸出PBS尽可能不完全干燥。加入50μL设置安装媒体（我们使用延长黄金），轻轻地放在封面上组织上的滑。安装一夜之间在室温下储存的幻灯片设置允许在-80 ° C。 </li><li>镜检，我们使用一个Z驱动器与一个宽视场显微镜。我们使用20X空气目标（NA 0.75，计划复消色差透镜）或60X的石油目标（NA 1.4，计划复消色差透镜）和2微米，1微米的Z轴部分，涵盖了整个肌层。随后，对图像进行卷积使用Autoquant反卷积软件。另外，许多人都使用共聚焦显微镜，而不是类似的结果。 </li></ol> 3。 Cryosection准备和免疫<ol><li>将消化道，浆膜侧下来，平放在滤纸上的每​​个部分。切断周围组织的滤纸。放置在滤纸上的组织在4％多聚甲醛的PBS（我们到之前使用的密封小瓶PBS稀释32％多聚甲醛）（PFA）在室温下2小时。固定，可以在6 - 12孔板上取决于组织的大小，或1.5毫升离心管。如果板，封口膜，以尽量减少煤灰曝光。较长的固定时间，可能会导致抗原遮蔽，特别是对反套件ACK - 2抗体。 </li><li>删除PFA和地方组织从30％蔗糖的PBS。允许组织在4℃过夜下沉° C。 </li><li> 1:1的比例30％的蔗糖10月和1小时的平衡组织。然后装入组织垂直与华侨城填充胃肠道的纵向轴线应平行的cyrosections cryomold。研究不同基因型的小鼠或不同的待遇时，也可能是有用的安装到同一cryomold不同的小鼠的组织，以确保并行处理。干冰冻结，在-80℃冷冻存储。 </li><li>切割10微米的冰冻切片到幻灯片和储存在-80 ° C冰箱。我们通常切两节每张幻灯片。我们通常也执行一个cryosection幻灯片H＆E染色。 </li><li>对于免疫荧光法，先在室温下平衡15分钟的幻灯片。然后绘制使用PAP笔圈周围组织。 </li><li> 1小时与150μL封闭液（整个装载免疫相同）座。 </li><li>吸封​​闭液，加入​​150μL封闭液稀释的第一抗体，并在4℃孵育过夜彗星在湿盒。 </li><li>用PBS清洗5分钟2倍。 </li><li>孵育稀释的二级抗体在阻断缓冲液在室温下2小时。我们通常使用Alexa的488抗兔和Alexa 594抗鼠抗体。 </li><li>为15分钟，用PBS 3X洗净。 </li><li>吸不完全干燥组织的情况下滑动。添加下降的硬盘设置，安装用DAPI（我们使用延长黄金）和地方＃1.5盖玻片的媒体。在室温下过夜设置并储存在-80 ° C冰箱放置幻灯片。 </li><li>收购三联荧光图像使用的DAPI，FITC和德克萨斯红过滤器集。 </li></ol> 4。代表性的成果： 在这里，我们作为一个例子大肠。 H＆E蔗糖染色保护固定冻结部分为免疫显示一些工件一个参考石蜡节（图2），其中黄线demarks的圆形和纵向的肌肉层，并在绿色线demarks之间的肌间神经丛之间的边界黏膜和muscularlis。研究cryosection，从浆膜黏膜，你第一次遇到较薄的纵肌（LM）层较厚的环形肌（CM）层。本节应在纵肌的同时，使原子核的LM应有所拉长，而原子核的CM应小而圆。 LM和厘米之间的肌间神经丛神经元染色与PGP9.5。 PGP9.5也污渍整个CM的神经元突起。肌间ICC网络（ICC - MY）环绕的肌间神经丛和多极化细胞。在纵向的肌肉层，也有罕见的肌肉注射国际商会（ICC - IMS），双极细胞，肌肉并联运行。在圆形的肌肉层，有丰富的ICC - IMS也并行运行的环形肌和这种切割横截面的双极细胞。在肌层和粘膜下层交界处，粘膜下的国际刑事法院ICC - SMPS组成的网络是一个多极化的国际商会的单位网络。检查整个大肠免疫安装套件，应该想到遇到的ICC我的网络第一，然后的CM - IM的国际刑事法院，国际刑事法院的SMP网络，作为一个重点从盖玻片（Figure. 3） 。体积测量使用整个安装图像三维重建国际刑事法院的网络已经描述10。使用宽视场显微镜，有显着的平面可反褶积后删除（图3）在原始图像的荧光。不完全剥离的黏膜会导致高背景荧光。 ANO1是另一个国际刑事法院的标记和工具包的合作免疫ANO1显示完整的ICC染色（图4）重叠。 <img src="/files/ftp_upload/2802/2802fig1.jpg" alt="图1"/> 图1代表解剖小鼠胃肠道，重印许可13。 <img src="/files/ftp_upload/2802/2802fig2.jpg" alt="图2"/> 图2。）代表H＆E的鼠标大肠准备描述和参考石蜡包埋节冰冻切片一些收缩文物冰冻切片染色。黄线demarks肌间神经丛和蓝线分离粘膜肌层。二）代表包（红色，ACK - 2）和PGP9.5（绿色）用DAPI（蓝色）鼠标大肠counterstaining双重免疫。 LM：纵肌CM：环形肌。比例尺20微米。 <img src="/files/ftp_upload/2802/2802fig3.jpg" alt="图3"/> 图3。代表包（红色的ACK - 2）整个安装免疫三个焦平面同一领域，ICC我乘坐的飞机在LM / CM的边界，在CM的ICC - IM的飞机，国际刑事法院的SMP的飞机在CM /粘膜下层边界。原始图像和卷积图像显示。比例尺20微米。 <img src="/files/ftp_upload/2802/2802fig4.jpg" alt="图4"/> 图4。代表Ano1（绿色）和大肠包（红色，ACK - 2）双免疫。比例尺20微米。

<ol>
	<li>Sanders, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+case+for+interstitial+cells+of+Cajal+as+pacemakers+and+mediators+of+neurotransmission+in+the+gastrointestinal+tract.">A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract.</a> Gastroenterology. 111, 492-515 (1996).</li><li>Ward, S. M., Sanders, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+and+pathophysiology+of+the+interstitial+cell+of+Cajal:+from+bench+to+bedside.+I.+Functional+development+and+plasticity+of+interstitial+cells+of+Cajal+networks.">Physiology and pathophysiology of the interstitial cell of Cajal: from bench to bedside. I. Functional development and plasticity of interstitial cells of Cajal networks.</a> Am J Physiol Gastrointest Liver Physiol. 281, 602-611 (2001).</li><li>Komuro, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+organization+of+interstitial+cells+of+Cajal+in+the+gastrointestinal+tract.">Structure and organization of interstitial cells of Cajal in the gastrointestinal tract.</a> J Physiol. 576, 653-658 (2006).</li><li>Sanders, K. M., Ordog, T., Koh, S. D., Torihashi, S., Ward, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+plasticity+of+interstitial+cells+of+Cajal.">Development and plasticity of interstitial cells of Cajal.</a> Neurogastroenterol Motil. 11, 311-338 (1999).</li><li>Ward, S. M., Burns, A. J., Torihashi, S., Sanders, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+of+the+proto-oncogene+c-kit+blocks+development+of+interstitial+cells+and+electrical+rhythmicity+in+murine+intestine.">Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine.</a> J Physiol. 480, 91-97 (1994).</li><li>Huizinga, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=W/kit+gene+required+for+interstitial+cells+of+Cajal+and+for+intestinal+pacemaker+activity.">W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity.</a> Nature. 373, 347-349 (1995).</li><li>Rubin, B. P., Heinrich, M. C., Corless, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+stromal+tumour.">Gastrointestinal stromal tumour.</a> Lancet. 369, 1731-1741 (2007).</li><li>Hirota, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain-of-function+mutations+of+c-kit+in+human+gastrointestinal+stromal+tumors.">Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors.</a> Science. 279, 577-580 (1998).</li><li>Chi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ETV1+is+a+lineage-specific+survival+factor+in+Gastrointestinal+Stromal+Tumour+(GIST).">ETV1 is a lineage-specific survival factor in Gastrointestinal Stromal Tumour (GIST).</a> Nature. , (2010).</li><li>Kwon, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+structure+and+function+of+ICC+networks+in+ICC+hyperplasia+and+gastrointestinal+stromal+tumors.">Changes in the structure and function of ICC networks in ICC hyperplasia and gastrointestinal stromal tumors.</a> Gastroenterology. 136, 630-639 (2009).</li><li>Hwang, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+anoctamin+1/TMEM16A+by+interstitial+cells+of+Cajal+is+fundamental+for+slow+wave+activity+in+gastrointestinal+muscles.">Expression of anoctamin 1/TMEM16A by interstitial cells of Cajal is fundamental for slow wave activity in gastrointestinal muscles.</a> J Physiol. 587, 4887-4904 (2009).</li><li>Gomez-Pinilla, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ano1+is+a+selective+marker+of+interstitial+cells+of+Cajal+in+the+human+and+mouse+gastrointestinal+tract.">Ano1 is a selective marker of interstitial cells of Cajal in the human and mouse gastrointestinal tract.</a> Am J Physiol Gastrointest Liver Physiol. 296, G1370-G1381 (2009).</li><li>Sommer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+stromal+tumors+in+a+mouse+model+by+targeted+mutation+of+the+Kit+receptor+tyrosine+kinase.">Gastrointestinal stromal tumors in a mouse model by targeted mutation of the Kit receptor tyrosine kinase.</a> Proc Natl Acad Sci U S A. 100, 6706-6711 (2003).</li></ol>

Cajal间质细胞的最初特点圣地亚哥Ramóón Ÿ卡哈尔整整一个世纪前用亚甲基蓝和银的消化道肌层铬酸污渍。卡哈尔最初认为，国际刑事法院是根据其流程的神经元轴突和树突让人想起。许多随后的几年里，国际刑事法院的生物学研究已经有限，缺乏特异性标志物，直到发现该试剂盒不仅是在国际刑事法院表示，但也为他们的发展6需要。 KIT免疫从那时起，已广泛应用于在国际刑事法院的生物学研究，也带动了其他如膀胱收缩机关升值的国际刑事法院。近日，ANO1已被确定为第二个可靠的国际刑事法院的标记。 在过去的15年，用免疫荧光法，以确定国际刑事法院使用各种固定和安装技术，已经有无数的出版物。在我们手中“固定冻结”已与多聚甲醛和蔗糖的保护前缀的冰冻切片最好的相比，丙酮，多聚甲醛后cryosectioning后固定。对于整个坐骑，我们发现，丙酮固定黏膜剥离给出了最强大的结果。 从历史上看，ACK - 2鼠标ICC识别已选择套件抗体。它是一只老鼠单克隆承认的胞外域，它也是一种封闭抗体，导致肠梗阻时，给予活体小鼠。然而，ACK - 2的抗原表位，正在慢慢摧毁了多聚甲醛固定和不标准的抗原检索方法救出。我们已经发现，ACK - 4表位的是更耐多聚甲醛固定。相比固定的冻块和切片，石蜡包埋块和部分保留形态更好，更适合长期归档（图4）。既不ACK - 2也不ACK - 4已成功地用于石蜡切片。我们有特点，D13A2，一个新的兔单克隆抗体，工作非常出色的消化道固定冷冻和石蜡切片。 套件是对一些其他类型的细胞，包括黑色素细胞，造血干细胞，生殖细胞，特别是肥大细胞在消化道中发现。幸运的是，大多数的肥大细胞被发现的黏膜和肌层。双兔抗Ano1和鼠抗包染色，可以消除每个抗体的非特异性染色（图4）。这也是值得注意的套件和Ano1国际刑事法院的子类之间的相对表现似乎有所不同。例如，在小肠，包染色肌间国际刑事法院的激烈得多相比，深肌丛ICC，而Ano1染色可比。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>鼠抗试剂盒抗体（克隆ACK - 2） </td><td> eBioscience公司</td><td> 14-1172 </td><td>在2μg/ml使用。抗原表位的是失去了与overfixation。 </td></tr><tr><td>鼠抗试剂盒抗体（克隆ACK - 4） </td><td> Cedarlane </td><td> CL8936AP </td><td>在2μg/ml使用。 </td></tr><tr><td>兔抗- KIT抗体（克隆D13A2） </td><td>细胞信号转导</td><td> 3074 </td><td>在1:100稀释使用。这里列出的只有抗体，石蜡切片。 </td></tr><tr><td>兔抗PGP9.5抗体</td><td> Abcam公司</td><td> ab10404 </td><td>标签的神经元细胞的细胞质。标记的肌间神经丛的神经元。 1:1000稀释使用。 </td></tr><tr><td>兔抗Ano1抗体</td><td> Abcam公司</td><td> ab53212 </td><td>使用在2μg/ml </td></tr><tr><td>的Alexa Fluor 594山羊抗鼠IgG </td><td> Invitrogen公司</td><td> A - 11007 </td><td>使用在2μg/ml </td></tr><tr><td>的Alexa Fluor 488山羊抗兔IgG </td><td> Invitrogen公司</td><td> A - 11008 </td><td>使用在2μg/ml </td></tr><tr><td>动物饲养针</td><td>费舍尔</td><td> 01-208-87 </td><td>硅放倒针冲洗成年小鼠胃肠道</td></tr><tr><td>钝针</td><td>流式细胞Dickison </td><td> 305180 </td><td>钝针可用于冲厕前断奶仔兔的胃肠道</td></tr><tr><td>用DAPI延长黄金的抗淬灭剂</td><td> Invitrogen公司</td><td> P - 36931 </td><td>可以使用替代硬盘设置安装用DAPI媒体</td></tr><tr><td>组织- TEK Cryomold </td><td>组织Tek的</td><td> 4557 </td><td></td></tr><tr><td>组织- TEK华侨城</td><td>组织Tek的</td><td> 4583 </td><td></td></tr><tr><td> 32％的多聚甲醛（10毫升密封安瓿） </td><td>电磁科学院</td><td> 15714 </td><td>使用前打开密封的安瓿和稀释至4％ </td></tr><tr><td>封闭液</td><td></td><td></td><td> 5％山羊血清，0.1％的Triton X - 100的PBS </td></tr></tbody></table>

visualization of the interstitial cells of cajal (icc) network in mice

（ICC）的卡哈尔间质细胞间质衍生的“起搏细胞”的胃肠道（GI）道产生蠕动所需的自发慢波和调解神经元的输入，从肠道的神经系统1。国际刑事法院的不同亚型形成不同的网络， 在消化道2,3肌层。损失或伤害，这些网络是与运动障碍4。 ICC细胞表达质膜和试剂盒已在过去的15年使用免疫标签ICC 网络 5,6 kit受体酪氨酸激酶。更重要的是，正常的Kit活动是5,6为国际刑事法院的发展需要。国际刑事法院的细胞在胃肠道间质瘤（GIST）的结果，即经常海港增益功能KIT突变7,8恶性转化。我们最近表明ETV1是一个谱系特异性生存率的因素，在国际刑事法院/ GIST的血统表示是主转录调节为正常的ICC网络的形成和胃肠间质肿瘤的发生9。我们进一步证明它与合作激活KIT突变在肿瘤的发生。在这里，我们描述了小鼠在国 ​​际刑事法院的网络可视化的方法，主要是基于对先前公布的协议10,11。最近anoctamin 1（ANO1），氯离子通道也被定性为一个特定的膜标记，国际刑事法院11,12。由于其质膜本地化，这两个蛋白的免疫可用于可视化的国际刑事法院的网络。在这里，我们描述了国际刑事法院的网络可视化固定冷冻cyrosections整装准备。

Watch this Scientific Journal Video about Visualization of the Interstitial Cells of Cajal ICC Network in Mice at JoVE.com

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

Cajal间的间质细胞（ICC）的胃肠道（GI）的起搏细胞。他们之间的平滑肌细胞和调节胃肠道收缩后神经节神经元纤维形成的复杂的网络。在这里，我们目前的横断面和整个安装小鼠ICC网络的可视化的免疫方法。

卡哈尔（ICC）的网络的间质细胞在小鼠体内的可视化

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

visualization-of-the-interstitial-cells-of-cajal-icc-network-in-mice

Research

JoVE Journal

Biology

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

54.2K 次观看.  Memorial Sloan Kettering Cancer Center. Cajal间的间质细胞（ICC）的胃肠道（GI）的起搏细胞。他们之间的平滑肌细胞和调节胃肠道收缩后神经节神经元纤维形成的复杂的网络。在这里，我们目前的横断面和整个安装小鼠ICC网络的可视化的免疫方法。

视频: Visualization of the Interstitial Cells of Cajal ICC Network in Mice

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

一个现场的固定化酵母细胞可视化的快速技术

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

科研

生物学

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

计数和确定培养细胞的可行性

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Trypsinizing和传代的哺乳动物细胞

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

Freezing, Thawing, and Packaging Cells for Transport

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks. 

Cultured mammalian cells are used extensively in cell biology studies. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks.

冷冻，解冻，和运输包装细胞

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

微阵列分析FOR酿酒酵母</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

在哺乳动物细胞中的内质网定位的mRNA的可视化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

内质网子域的培养细胞中的可视化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly and fate is important to understand the cellular response to stress. The deficiency in key factors of SGs like G3BP (RasGAP SH3 domain Binding Protein) leads to developmental defects in mice&#160;and alterations of the Central Nervous System. To study the dynamics of SGs in cells from an organism, one can culture primary cells and follow the localization of a transfected tagged component of SGs. We describe time-lapse experiment to observe G3BP1-containing SGs in Mouse Embryonic Fibroblasts (MEFs). This technique can also be used to study G3BP-containing SGs in live neurons, which is crucial as it was recently shown that these SGs are formed at the onset of neurodegenerative diseases like Alzheimer&#39;s disease. This approach can be adapted to any other cellular body and granule protein component, and performed with transgenic animals, allowing the live study of granules dynamics for example in the absence of a specific factor of these granules.

Stress granules (SGs) are cytoplasmic RNA granules containing stalled ribonucleoprotein particles (RNPs), and important in cellular response to various stresses. Dynamics of SGs can be followed in live cells by visualizing the localization of a tagged component of SGs in transfected primary cells after stress.

中G3BP应力颗粒动力学在Live原代细胞可视化

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly ...