The authors would like to acknowledge Wensheng Liu for invaluable help with cell imaging. This work was supported by grants from the&#160;US National Institutes of Health (R01 CA182467), American Cancer Society (RSG-09-252-01-RMC), Lynn Sage Foundation, and A Sister&#8217;s Hope Foundation.

EMT诱导1细胞培养注：EMT可以通过治疗TGFβ或转录因子扭，蜗牛，或ZEB1 / 2在上皮细胞异常表达所诱导。另外，在本协议是通过扭ER融合蛋白在人永生化乳腺上皮细胞（HMLE /扭ER，J杨博士的礼物，加州大学圣地亚哥分校）9,11,29表达可诱导的EMT系统。当4-羟基他莫昔（TAM）的治疗中，融合蛋白的扭ER转位到细胞核中以驱动转录并产生一个完整的EMT的过渡在12-14天9,11,29。额外的EMT诱导系统，如HMLE /蜗牛-ER，HMLE / TGF-β，和MCF10A / TGF-β，可在我们以前的出版物11,30中找到。 <ol><li>在37℃的培养箱中，用5％的CO 2保持HMLE /扭ER的细胞在无血清乳腺上皮细胞生长培养基（MEGM）。通过细胞每2-3天，当他们达到80％汇合。孵育的细胞用0.15％胰蛋白酶在37℃下进行5-10分钟，然后失活胰蛋白酶，用5％小牛血清/ DMEM中。降速细胞和悬浮他们MEGM。板的细胞在新的组织培养皿中，在1〜4的比例。 </li><li>谭溶解在乙醇，使200微米的解决方案。从光存储保护TAM在-20℃。使用前，刚加谭成MEGM媒体在1：10,000稀释，使20纳米谭终浓度。 </li><li>对于诱导EMT的，板材1.6×10 6 HMLE /扭ER细胞变成10厘米的组织培养皿，一式两份。培养细胞与20纳米含谭MEGM媒体。 </li><li>板1.6×10 6个HMLE /扭ER细胞分化成一式两份的10cm平皿和治疗的细胞用0.01％的乙醇作为非TAM处理的控制。 </li><li>孵化两天后，拿在光学显微镜下的照片在放大10倍来记录细胞的形态变化。 </li><li>从控制或TAM治疗的一盘吸出培养基细胞。由报废一半的板块中的RNA裂解缓冲液中分离RNA并在RIPA缓冲液，另一半用于蛋白质分析洗涤细胞用冷的PBS中，并收获细胞。 </li><li>传代细胞，从在一式两份的10cm平皿的控制或TAM处理的菜肴由再镀1.6×10 6个细胞。连续处理的细胞用20nM的TAM或车辆。 </li><li>重复步骤5到7隔日一次，直到第14天，在此期间，TAM处理的扭ER的细胞表现出纺锤形的间充质的形态，而载体处理的对照细胞维持鹅卵石状上皮形态。 </li></ol> EMT的表征2。 注：完成EMT是通过表明：（1）细胞从鹅卵石样上皮细胞形态为梭形纤维母细胞样外观形态变化; （2）E-cadherin的定位在细胞与细胞连接处的损失;的EMT标志物和（3）转换表达式，由损耗定义上皮标记和间质标记物的增益。 细胞的形态变化是通过光学显微镜在EMT诱导的时间过程中放大10倍拍摄的，在本节中描述了第一节免疫荧光检测E-钙粘着蛋白在细胞结的说明。的EMT标志物的表达通过免疫印迹法检测。常见的上皮标记为E-钙粘蛋白，γ-catenin及occludin的，和间质标记物包括纤维连接蛋白，N-钙粘蛋白和波形。免疫印迹法的一般过程在第4节中描述。 <ol><li>谭处理，种子1×10 5细胞在12毫米的玻璃圆形盖玻片置于24孔板的孔的第12天。 </li><li>播种，吸中，洗涤细胞，预热的PBS后48小时。 </li><li>固定的细胞用预热的4％多聚甲醛固定15分钟。 </li><li>用PBS洗涤细胞三次。 </li><li>孵育的细胞用0.2％的Triton X-100的PBS中的15分在室温。 </li><li>用PBS洗涤细胞三次。 </li><li>孵育细胞在5％BSA / PBS进行1小时，在室温到阻断非特异性结合。 </li><li>孵育细胞与第一抗体的E-钙粘蛋白在1:50稀释1％BSA / PBS在湿盒在o / n 4℃。注：范围为初级抗体的稀释通常是1:50〜1：200，并为每个抗体的最佳稀释度应通过试验确定。 </li><li>滗出第一抗体并用PBS洗涤细胞三次。 </li><li>孵育在1细胞用荧光标记的二抗：500稀释为1小时，在室温。从在此步骤中，保护细胞免受光。 </li><li>滗二次抗体和洗三次，用PBS。 </li><li>染色的细胞用0.1〜1克/毫升的DAPI或Hoechst的10分钟。 </li><li>漂洗细胞，用PBS三次。 </li><li>摩盖玻片用一滴安装介质，并密封盖玻片指甲油。 </li><li>观察细胞，并采取à63X荧光显微镜下的图像。 </li></ol>用另外的抗体染色，可以执行确认EMT表型。例如，N-cadherin和α-平滑肌肌动蛋白可染的骨髓间充质表型29,31,32。细胞骨架结构的重组可以通过与鬼笔环肽的F-肌动蛋白11染色细胞进行监控。此外，测定对细胞运动性和细胞死亡的电阻可以被执行以检查EMT 11的属性。 利用定量RT-PCR检测3剪接异构体的检测<ol><li>引物设计 注：引物对，应精心设计，放大不同的剪接异构体。外显子跳跃，最常观察到的选择性剪接事件，在此示为一个例子来说明引物设计的策略。 图1A示出了四个外显子的mRNA前体，与所述第三外显子的可变外显子。纳入外显子3的结果生产更长的剪接异构体，而跳过外显子3的产生较短的剪接异构体。引物需要被设计以区分外显子3，包括同种型和外显子3跳过的同种型，并检测该mRNA的总转录物。 <ol><li>用于检测外显子包含的，精心设计的可变外显子3，并在附近的组成型外显子4的内侧的反向引物内的正向引物，允许可变外显子包含同种型的特异性扩增。确保不设计正向和反向的相同变量的外显子中的引物，以避免从基因组DNA或mRNA前体的PCR扩增产物。 </li><li>特异性扩增外显子跳跃事件，设计的横跨构外显子2和外显子4，当可变外显子3被包括，否则将被破坏的交界处的引物之一。设计构成的外显子4，这样里面的另一个引物，PCR产物生成，只有当可变外显子3 exclud埃德和本构外显子2和外显子4随后结合在一起。 </li><li>以检测该基因的总转录物，设计中的两个构个外显子1和2的引物，因此，PCR产物是从所有亚型扩增。 </li><li>确保熔化温度（Tm）为所有的引物是约55℃时，各引物的长度是约18-22个核苷酸，PCR产物的大小为基础对介于80和150。 </li></ol></li><li> RNA提取：提取的RNA使用总RNA提取试剂盒（见表材料）。 <ol><li>收集细胞350μL的RNA裂解液。 </li><li>加入350微升70％乙醇的裂解物并充分混合。 </li><li>将样品转移到RNA的纯化塔。 </li><li>离心机以10,000 xg离心1分钟，丢弃流过。 </li><li>用RNA洗涤缓冲液洗二柱曾用500微升的RNA洗涤缓冲液I和的两倍。 </li><li>去除残留的RNA洗涤缓冲液II通过离心从在最大速度下的柱2分钟。 </li><li>柱转移至新的1.5毫升试管中，加入50μl的无核酸酶的水在柱基质的中心，并通过离心分离以最大速度洗脱RNA。 </li></ol></li><li>第一链cDNA的合成<ol><li>确定浓度和RNA的质量用紫外吸收测量。注：质量好的RNA应该有大约2.0 260纳米/ 280纳米的吸收率。 </li><li>设置逆转录酶（RT）反应包含250ng的总RNA，0.05微克随机六聚体引物，50皮摩尔的MgCl 2，10皮摩尔的dNTP，2微升5×GoScript缓冲液中，加入1μl逆转录酶在20微升的最终体积。 </li><li>进行RT反应在42℃进行1小时，接着温育在70℃下5分钟，以灭活逆转录酶。 </li></ol></li><li>实时荧光定量PCR <ol><li>成立20微升反应，由10微升的SYBR Green预混液，0.1-1.0微升cDNA模板，和5皮摩尔的正向和反向引物。 </li><li>运行实时PCR用下列步骤进行40个循环：95℃变性10秒，58℃退火10秒，和60℃延伸30秒。一式三份进行PCR反应。 </li><li>检查解离曲线，并确保具有单峰被观测到的每个引物组。 </li><li>通过计算扩增曲线的二阶导数的值获得的量化周期（Cq的）值。 </li><li>相比，使用“增量增量Cq的（ΔΔCq）&#39;所示的由下述式方法的未诱导的样品计算诱导样品中的靶mRNA的相对量（RQ）。使用管家基因，如GAPDH或TBP，作为参考。 </li></ol></li></ol><img alt="式（1）" fo:content-width="3in" src="/files/ftp_upload/51845/51845eq1.jpg" /> 为了更准确地计算剪接异构体的相对值，叔他使​​用多个参考基因应该被考虑。结果，能使用由Pfaffl 等人 33,34所述的改性绝对值量化方法进行分析。 在蛋白质水平考试4剪接异构体的<ol><li>收集细胞在100μl冰冷的RIPA缓冲液。 </li><li>设置在冰上裂解物为约10分钟。 </li><li>离心机以14,000 xg离心10分钟，在4℃，并收集上清液。 </li><li>测定蛋白浓度用Bradford检测。 </li><li>稀释在SDS上样缓冲液的蛋白质样品并装载20-40微克蛋白质在10％SDS-PAGE凝胶。 </li><li>跑SDS-PAGE凝胶在20mA 1.5小时。 </li><li>根据靶蛋白的分子量转移蛋白至PVDF膜在湿的传送系统，在4℃下进行3小时，在85 V.调整传输时间。 </li><li>块膜在5％脱脂乳进行30分钟至1小时，在室温下。 </li><li>孵育膜无线TH一抗，4℃CO / N。的范围为初级抗体的稀释通常是在1：200〜1：5000，确定每个抗体实验的最佳稀释度。在这个协议中使用的抗体的稀释可以在“表特定的试剂和设备”中找到。 <ol><li>为了检测剪接异构体，使用识别特定亚型的特异性抗体。另外，使用该识别出组成型外显子编码区的抗原决定簇的抗体，同时检测剪接异构体根据其不同的蛋白质的大小。 </li><li>在同一时间，通过免疫印迹对EMT标志物监测EMT。使用E-钙粘蛋白，γ-catenin及occludin的上皮标记和纤维连接蛋白，N-钙粘蛋白和波形间充质标记。 </li></ol></li><li>洗膜三次，用TBS-T（50毫摩尔Tris，150 mM氯化钠，0.05％吐温20，pH 7.4）中，在5分钟的时间间隔。 </li><li>孵育膜与HRP偶联的二级蚂蚁ibody以1:10,000稀释为1小时，在室温。 </li><li>洗膜3次用TBS-T以5分钟的间隔。 </li><li>可视化蛋白用化学发光检测系统和所述膜暴露于放射自显影胶片。 </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

这里描述的方法使得能够选择性剪接的检测中可诱导的EMT模型。如剪接同种型的表达，例如，动态改变可以在整个EMT的时间过程被捕获。这种方法比使用不同的上皮 - 间质或，代表细胞系的选择性剪接的比较优势，因为从联合国有关的细胞不同的遗传背景，可能不适当地影响选择性剪接。然而，成功诱导的EMT必须谨慎使用上的变化剪接任何结论之前，各种EMT的标志，可以得出证实。此外，除了此?...

上皮 - 间质转化（EMT）是推动胚胎发育过程中的器官形态和组织重塑一个发展计划。当异常激活，EMT促进肿瘤转移和器官纤维化1,2。 EMT过程中引人注目的研究已经描述转录调控的重要性，通过几个转录因子，如扭曲，蜗牛和ZEB，这抑制了粘着连接蛋白的E-钙粘蛋白的表达，从而导致cobble-的损失定义石样上皮形态学和纺锤状的间充质表型3-8的增益。通过RNA的全基因组分析，最近的研究表明，存在一组基因的剪接方式有两种上皮或间质表型9,10相关联的。从我们的实验室工作功能连接选择性剪接和EMT。通过研究细胞表面粘附分子CD44，我们证明了CD44 ALTERNative剪接EMT过程中紧密调节，并且更重要的是，CD44剪接同种型转换因果有助于EMT 11。 选择性剪接表示基因调控的一种普遍的和保守的模型，如高达95％的人的多外显子基因可变剪接12-14。通过从单个基因产生多个蛋白的产品，选择性剪接构成蛋白多样性的重要机制，增加了另一层复杂性，以人类基因组。因此，选择性剪接的失调可能会导致引起人类疾病的深刻的生物学效应。事实上，在疾病的异常剪接都记载了十多年15-25，其中包括在基因编码的剪接突变机械骨髓增生异常综合征26-28通常发现最近的调查结果。因此，开发可靠的方法的选择性剪接í检测soforms是在不同的生物过程，包括EMT的研究非常重要的。 在这里，我们提供了一个协议来检测使用可诱导的EMT模型的变化剪接。该方法用于设计PCR引物和检测剪接异构体是合适的，不仅用于选择性剪接的EMT过程中的研究，而且对选择性剪接在其他生物过程的研究。 EMT过程中调查剪接是必须为了更好地理解EMT和肿瘤转移的机制，从而促进有效的策略的开发来治疗癌症转移。

<table border="0" cellpadding="0" cellspacing="0" width="1167">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Name of the reagent</td>
			<td style="width:183px;">Company</td>
			<td style="width:123px;">Catalogue number</td>
			<td style="width:564px;">Comments (optional)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-hydroxytamoxifen</td>
			<td>Sigma</td>
			<td>H7904</td>
			<td>Make stock solution by dissolving in ethanol to 200&#956;M, and keep at -20&#8451; protected from light.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E.Z.N.A. Total RNA Isolation kit</td>
			<td>Omega Bio-Tek</td>
			<td>R6731</td>
			<td>Total RNA isolation kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GoScript Reverse Transcription System</td>
			<td>Promega</td>
			<td>A5001</td>
			<td>Reagent for qRT-PCR assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GoTaq qPCR Master Mix</td>
			<td>Promega</td>
			<td>A6002</td>
			<td>Reagent for qRT-PCR assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LightCycler 480 Real-Time PCR System</td>
			<td>Roche</td>
			<td></td>
			<td>Equipment for qRT-PCR assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD44 antibody</td>
			<td>R&#38;D Systems</td>
			<td>BBA10</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E-cadherin antibody</td>
			<td>Cell Signaling Technology</td>
			<td>4065</td>
			<td>1:2500 dilution for immunoblotting; 1:50 dilution for immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#947;-catenin antibody</td>
			<td>Cell Signaling Technology</td>
			<td>2309</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">occludin antibody</td>
			<td>Santa Cruz Biotechnology Inc.</td>
			<td>sc-5562</td>
			<td>1:500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fibronectin antibody</td>
			<td>BD Transduction Laboratories</td>
			<td>610077</td>
			<td>1:5000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-cadherin antibody</td>
			<td>BD Transduction Laboratories</td>
			<td>610920</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vimentin antibody</td>
			<td>NeoMarkers</td>
			<td>MS-129-p1</td>
			<td>1:500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GAPDH antibody</td>
			<td>Millipore Corporation</td>
			<td>MAB374</td>
			<td>1:10000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amasham ECL Western blotting detection reagent</td>
			<td>GE Health Life Science</td>
			<td>RPN2209</td>
			<td>Chemiluminescence system</td>
		</tr>
	</tbody>
</table>

detection of alternative splicing during epithelial-mesenchymal transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

上述过程提供了一个可靠的方法EMT过程中检测可变剪接。的CD44剪接同种型转换时扭诱导的EMT代表性的结果在下面给出的一个例子。 在HMLE /扭ER细胞扭诱导的EMT的特点是从鹅卵石状上皮细胞表型的细长成纤维细胞表型的转变（ 图2A），无上皮标志物E-钙粘蛋白，γ-catenin和occludin和中上调的间质标记物纤连蛋白，N-钙粘蛋白，波形蛋白（ 图2B）。此外，EMT...

Watch this Scientific Journal Video about Detection of Alternative Splicing During Epithelial-Mesenchymal Transition at JoVE.com

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

检测选择性剪接的过程中上皮 - 间质转化

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various ...

detection-alternative-splicing-during-epithelial-mesenchymal

Research

JoVE Journal

Biology

12.8K 次观看.  Northwestern University Feinberg School of Medicine. Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

视频: Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

光/暗转换测试小鼠

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

科研

生物学

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

蛋白泛素化的检测

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

优化的基于PCR的检测支原体

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

另类文化的人多能干细胞的生产，维护和遗传分析

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually untranslated &#8212; first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

方法发现小鼠BCRP1的另类用法子和转录调控

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

对于样品制备与TEM分析低细胞数量的另一种方法

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&amp;#946; Signaling

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

转化生长因子β信号诱导内皮-间质转移的分子分析

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...