Name: an immunofluorescent method for characterization of barrett&#8217;s esophagus cells
Uploaded: 2014-07-20T15:00:00
Description: Watch this Scientific Journal Video about An Immunofluorescent Method for Characterization of BarrettÃ¢â‚¬â„¢s Esophagus Cells at JoVE.com

这项工作是从圣，若瑟基金会（AJF，LJI）和美国肺脏协会，RG-224607-N（LJI）资助。

1。细胞株维护<ol><li>永生化人是高度不典型增生（CP-B，CP-C，CP-D）和化生（CP-）细胞株的端粒酶稳定转染逆转录酶（TERT）蛋白11。 </li><li>维持在补充有牛垂体提取物（BPE），表皮生长因子（EGF），5％胎牛血清（FBS），非必需氨基酸（NEAA），青霉素 - 链霉素 - 新霉素（PSN）角质形成细胞介质的细胞系，以及标准组织培养条件下（37℃，5％CO 2，98％湿度）。 </li></ol><p class="jove_titl...

<ol>
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Surg. 145, 531-538 (2013).</li><li>Jovov, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+transport+and+barrier+function+in+a+telomerase-immortalized+human+nondysplastic,+Barrett's+cell+line+(BAR-T).">Ion transport and barrier function in a telomerase-immortalized human nondysplastic, Barrett's cell line (BAR-T).</a> Dis. Esophagus. 22, 386-395 (2009).</li><li>Merlo, L. M., Kosoff, R. E., Gardiner, K. L., Maley, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+co-culture+model+of+esophageal+cells+identifies+ascorbic+acid+as+a+modulator+of+cell+competition.">An in vitro co-culture model of esophageal cells identifies ascorbic acid as a modulator of cell competition.</a> BMC Cancer. 11, 461 (2011).</li><li>Zhang, H. Y., Hormi-Carver, K., Zhang, X., Spechler, S. J., Souza, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+benign+Barrett's+epithelial+cells,+acid+exposure+generates+reactive+oxygen+species+that+cause+DNA+double-strand+breaks.">In benign Barrett's epithelial cells, acid exposure generates reactive oxygen species that cause DNA double-strand breaks.</a> Cancer Res. 69, 9083-9089 (2009).</li><li>Palanca-Wessels, M. C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extended+lifespan+of+Barrett's+esophagus+epithelium+transduced+with+the+human+telomerase+catalytic+subunit:+a+useful+in+vitro+model.">Extended lifespan of Barrett's esophagus epithelium transduced with the human telomerase catalytic subunit: a useful in vitro model.</a> Carcinogenesis. 24, 1183-1190 (2003).</li><li>Kumble, S., Omary, M. B., Cartwright, C. A., Triadafilopoulos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Src+activation+in+malignant+and+premalignant+epithelia+of+Barrett's+esophagus.">Src activation in malignant and premalignant epithelia of Barrett's esophagus.</a> Gastroenterology. 112, 348-356 (1997).</li><li>Inge, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha-Catenin+overrides+Src-dependent+activation+of+beta-catenin+oncogenic+signaling.">alpha-Catenin overrides Src-dependent activation of beta-catenin oncogenic signaling.</a> Mol. Cancer Ther. 7, 1386-1397 (2008).</li><li>Coluccia, A. M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SKI-606+Decreases+Growth+and+Motility+of+Colorectal+Cancer+Cells+by+Preventing+pp60(c-Src)-Dependent+Tyrosine+Phosphorylation+of+&#946;-Catenin+and+Its+Nuclear+Signaling.">SKI-606 Decreases Growth and Motility of Colorectal Cancer Cells by Preventing pp60(c-Src)-Dependent Tyrosine Phosphorylation of &#946;-Catenin and Its Nuclear Signaling.</a> Cancer Res. 66, 2279-2286 (2006).</li><li>Nam, J. -. S., Ino, Y., Sakamoto, M., Hirohashi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Src+Family+Kinase+Inhibitor+PP2+Restores+the+E-Cadherin/Catenin+Cell+Adhesion+System+in+Human+Cancer+Cells+and+Reduces+Cancer+Metastasis.">Src Family Kinase Inhibitor PP2 Restores the E-Cadherin/Catenin Cell Adhesion System in Human Cancer Cells and Reduces Cancer Metastasis.</a> Clin. Cancer Res. 8, 2430-2436 (2002).</li><li>Chu, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p27+phosphorylation+by+Src+regulates+inhibition+of+cyclin+E-Cdk2.">p27 phosphorylation by Src regulates inhibition of cyclin E-Cdk2.</a> Cell. 128, 281-294 (2007).</li><li>Inge, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+E-cadherin+promotes+cell+survival+by+activating+epidermal+growth+factor+receptor.">Soluble E-cadherin promotes cell survival by activating epidermal growth factor receptor.</a> Exp. Res. 317, 838-848 (2011).</li><li>Gan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+roles+of+ERK+and+Akt+pathways+in+regulation+of+EGFR-mediated+signaling+and+motility+in+prostate+cancer+cells.">Differential roles of ERK and Akt pathways in regulation of EGFR-mediated signaling and motility in prostate cancer cells.</a> Oncogene. 29, 4947-4958 (2010).</li></ol>

我们提出的申请，如果来的细胞对阐明在这些细胞的靶向治疗药物的生理效应的方法。尽管我们已经描述了使用对BE细胞这些过程，我们已经发现，这些方法也适用于各种不同类型的细胞13,17,18的。此外，这些程序可以改变多种方式来优化染色的具体目标。 我们发现，在具有适当的中频，直接影响一个参数是固定的选择。我们已经描述了使用4％PFA / PBS中固定在上面的?...

巴雷特食管（BE）是在食管的正常鳞状上皮和慢性暴露于胃食管返流疾病（GERD）1得到的胃内容物的结果化生变化。 BE被认为是针对胃食管反流病的一种保护机制，不过需要的存在赋予食管腺癌（EAC），这种疾病它带有一个显著较差生存1的风险增加。目前的估计表明，美国人口最多5.6％的BE，但是因为BE是常无症状，它被认为是被大多数人仍然未确诊2。由于两个GERD和EAC的发病率已经看到了持续增长3，它已成为重要的是了解参与是EAC的发展的分子机制，特别是因为这些信息可能会提供对预防是选管会的进展的治疗方法。 1的当前模式。慢性炎症，损伤，以及长期暴露于胃内容物造成基因毒性损伤施加的BE病灶促进肿瘤进展到选管会选择压力。许多遗传​​改变已经确定过程中，以选管会的进展。然而，尽管这有一个明显的缺乏了解的细胞信号转导的确切变化，在细胞结构和功能后续影响。 永生体外细胞系是研究细胞信号传导的影响，特别是在调查针对性治疗化合物的作用的有用工具。 BE永生化细胞系的近期商业可用性允许这种研究。虽然高通量测定法，如广泛使用的活力检测，可以在评估靶向疗法后的细胞增殖和苏效果是有价值的rvival 4-6，这些测定是不用于询问细胞信号后的细胞形态的影响是有用的。免疫荧光染色（IF）是用于研究的靶向治疗后细胞的形态学，生长和存活的特性和所涉及的蛋白质的效果的有效技术。我们实验室已经适应了这些方法对永生化BE细胞系，用IF来评价在划定一个可能的化学预防治疗和生物标志物的药物治疗7的希望时BE细胞系在临床可用的药物的作用。同样，在选管会应用这些技术，BE和食管永生化细胞系已划定有关BE选管会的进展8-10关键的发现。我们发现如果用靶向疗法治疗BE细胞的分析具有价值，允许改变细胞结构和蛋白定位的表征。在这里，我们提出我们的方法，如果永生BE细胞的characterize药物治疗。

<table border="0" cellpadding="0" cellspacing="0" width="743">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Name of the Material/Equipment</td>
			<td style="width:116px;">Company</td>
			<td style="width:108px;">Catalog Number</td>
			<td style="width:224px;">Comments/ Description (optional)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CP-A (Metaplastic Cell Line)</td>
			<td>ATCC</td>
			<td>CRL-4027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CP-B (High-grade Dysplastic Cell Line)</td>
			<td>ATCC</td>
			<td>CRL-4028</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CP-C (High-grade Dysplastic Cell Line)</td>
			<td>ATCC</td>
			<td>CRL-4029</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CP-D (High-grade Dysplastic Cell Line)</td>
			<td>ATCC</td>
			<td>CRL-4030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Keratinocyte-SFM (1X), Liquid</td>
			<td>Life Technologies</td>
			<td>17005-042</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.25% Trypsin-EDTA (1X), Phenol Red&#160;</td>
			<td>Life Technologies</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum, Qualified, HI</td>
			<td>Life Technologies</td>
			<td>10438026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PSN ANTIBIOTIC MIXTURE</td>
			<td>Life Technologies</td>
			<td>15640</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS - Phosphate-Buffered Saline</td>
			<td>Life Technologies</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pro-long Gold Antifade Reagent with DAPI&#160;</td>
			<td>Life Technologies</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Circular Glass Coverslip 18mm</td>
			<td>Fisher Scientific</td>
			<td>15-183-86</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#946;-catenin Primary Antibody (Rabbit)</td>
			<td>Cell Signaling</td>
			<td>9562S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexafluor 488 (Goat Anti-Rabbit)</td>
			<td>Life Technologies</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10cm TC treated PS dish, sterile</td>
			<td>USA Scientific</td>
			<td>CC7682-3394</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">12-well TC treated PS plate, sterile</td>
			<td>USA Scientific</td>
			<td>5666-5180</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>276855</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Saponin</td>
			<td>Sigma-Aldrich</td>
			<td>47036</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Serum Albumin - Fraction V</td>
			<td>Sigma-Aldrich</td>
			<td>85040C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SKI-606 (Bosutinib)</td>
			<td>Selleck Chemicals</td>
			<td>S1014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Square Bioassay Dish&#160;</td>
			<td>Thermo Scientific</td>
			<td>240835</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Parafilm&#160;</td>
			<td>VWR</td>
			<td>82024-546</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Disposable Pasteur Pipets, Flint Glass</td>
			<td>VWR</td>
			<td>14672-380</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nexcelom Mini Cell Counter</td>
			<td>Nexcelom</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cellometer Counting Chambers</td>
			<td>Nexcelom</td>
			<td>CHT4-SD100-014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zeiss Apotome microscope&#160;</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an immunofluorescent method for characterization of barrett&#8217;s esophagus cells

食管腺癌（EAC）小于17％，总生存率和EAC的发病率急剧上升，在过去的二十年。其中选管会的主要危险因素是Barrett食管（BE），正常食管鳞状在应对慢性胃灼热化生变化。尽管东非共同体之间的行之有效的连接进行的分子事件，涉及的进展特别是改变信号通路的审讯是选管会，了解甚少。这在很大程度上是由于可用来研究这些疾病的体外模型缺乏合适的。最近，永生BE细胞系已成为市售允许在体外研究中BE的。在这里，我们提出了BE永生细胞株的免疫荧光染色的方法，使在细胞信号传导和结构暴露于治疗化合物后体外表征。这些技术的应用将HELp开发洞察参与是EAC的发展机制，并为治疗和预防EAC的潜在途径。

从应用程序的描述的方法获得的结果的一个例子示于图1A-D中 。盖玻片带CP-D和CP-C BE细胞24小时，分别用1微米的Src家族抑制剂，SKI-606，或车辆（DMSO），并使用了粘着连接和Wnt上述程序信号蛋白，β染色-catenin的。 β-连环蛋白的可视化是通过用抗兔IgG结合到绿色荧光标记来实现，而在细胞核的标记是完成了用DAPI（蓝色）标记。盖玻片封片使用硬设置安装并使用激光扫描共聚焦显微镜使?...

Watch this Scientific Journal Video about An Immunofluorescent Method for Characterization of BarrettÃ¢â‚¬â„¢s Esophagus Cells at JoVE.com

An Immunofluorescent Method for Characterization of BarrettÃƒÂ¢Ã¢â€šÂ¬Ã¢â€žÂ¢s Esophagus Cells

有一个明显的需要对Barrett食管的分子驱动程序改进的信息。免疫荧光染色对于理解细胞信号转导的对细胞形态的影响的有效技术。我们提出了一个简单，有效的协议，使用免疫荧光染色，以评估在Barrett食管细胞治疗治疗。

免疫荧光法Barrett食管细胞的表征

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Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

瑞士改进轧制技术肠组织准备免疫组化和免疫分析

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

An Easy Method for Plant Polysome Profiling

植物多聚核糖体剖面的简单方法

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

肌肉干细胞中自噬的原位免疫荧光染色

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...