Name: time-lapse imaging of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer
Uploaded: 2013-02-08T12:00:00
Description: Watch this Scientific Journal Video about Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer at JoVE.com

作者要感谢“博凡吴基督教Raithel的为他介绍活细胞成像技术援助和迈克尔·丽格。由国家癌症研究所，美国国立卫生研究院RO1CA112176（PAF），美国国立癌症研究所，美国国立卫生研究院支持。 R01CA89041-10S1（PAF），，丁斯特的EV A/09/72227编号德国学术Austaush。 316（REN），国防部W81XWH-11-1-0074（REN），WCU（世界一流大学）计划，通过教育，科学和技术部（R31-10069）由韩国国家研究基金会（PAF ），美国国立卫生研究院IG20 RR025828-01（啮齿动物屏障设施设备），和，NIH：NCI 5P30CA051008（显微镜和影像和动物共享资源）。

<html> 1。总体方案<ol><li>生成基因工程乳腺的癌前病变的乳腺上皮细胞的原代培养和控制野生型小鼠。 </li><li>捕捉活细胞图像，每15分钟使用Volocity图像采集软件（版本5.3.1，珀金埃尔默，位于Waltham，MA）长达5天。 </li><li>查看时间推移图像直接评估的时间和上皮细胞的集落形成机制，细胞凋亡的发生率，并逐步形态的变化。 </li><li> Volocity产生。TIFF堆栈转换的JPG文件使用的MetaMorph的显微镜自动化?...

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	<li>Jones, L. P., 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=Activation+of+estrogen+signaling+pathways+collaborates+with+loss+of+Brca1+to+promote+development+of+ERalpha-negative+and+ERalpha-positive+mammary+preneoplasia+and+cancer.">Activation of estrogen signaling pathways collaborates with loss of Brca1 to promote development of ERalpha-negative and ERalpha-positive mammary preneoplasia and cancer.</a> Oncogene. 27, 794-802 (2008).</li><li>Burga, L. N., 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=Altered+proliferation+and+differentiation+properties+of+primary+mammary+epithelial+cells+from+BRCA1+mutation+carriers.">Altered proliferation and differentiation properties of primary mammary epithelial cells from BRCA1 mutation carriers.</a> Cancer Res. 69, 1273-1278 (2009).</li><li>Li, M., Wagner, K., Furth, P. A., Ip, M., Asch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+primary+mammary+epithelial+cells+and+transfection+by+viral+and+nonviral+methods.">Preparation of primary mammary epithelial cells and transfection by viral and nonviral methods.</a> Methods in Mammary Gland Biology and Breast Cancer. , (2000).</li><li>Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+stem-cell-driven+regeneration+in+mammals.">Imaging stem-cell-driven regeneration in mammals.</a> Nature. 453, 345-351 (2008).</li><li>Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+single-cell+imaging+of+mammalian+stem+cells.">Long-term single-cell imaging of mammalian stem cells.</a> Nature Methods. 8, S30-S35 (2011).</li><li>Eilken, H. M., Nishikawa, S. -. I., Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+single-cell+imaging+of+blood+generation+from+haemogenic+endothelium.">Continuous single-cell imaging of blood generation from haemogenic endothelium.</a> Nature. 457, 896-900 (2009).</li><li>Kokkaliaris, K. D., Loeffler, D., Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+tracking+hematopoiesis+at+the+single-cell+level.">Advances in tracking hematopoiesis at the single-cell level.</a> Current opinion in hematology. , (2012).</li><li>Rieger, M. A., Hoppe, P. S., Smejkal, B. M., Eitelhuber, A. C., Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+cytokines+can+instruct+lineage+choice.">Hematopoietic cytokines can instruct lineage choice.</a> Science. 325, 217-218 (2009).</li><li>Rieger, M. A., Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instruction+of+lineage+choice+by+hematopoietic+cytokines.">Instruction of lineage choice by hematopoietic cytokines.</a> Cell Cycle. 8, 4019-4020 (2009).</li><li>Heinrich, C., 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=Generation+of+subtype-specific+neurons+from+postnatal+astroglia+of+the+mouse+cerebral+cortex.">Generation of subtype-specific neurons from postnatal astroglia of the mouse cerebral cortex.</a> Nat. Protoc. 6, 214-228 (2011).</li></ol>

关键步骤重要的是要确保从小鼠同年龄控制在乳腺上皮细胞的行为与年龄有关的变化，乳腺腺体的收获。当镀敷的细胞，细胞的相同数目的应被镀在各孔中，为每一个实验。细胞应该是相对稀疏时，镀敷，使文化不成为汇合太快使得可以按照多个单独的细胞通过串行图像。至关重要的是，该板是在孵化器中平衡，因为焦点成像之前将更改后的板平衡。一次成像已经开始，应经常...

遗传工程小鼠模型的工具来研究和了解不同的遗传病变患乳腺癌的风险。例如，已经表明，遗传工程小鼠的三个因素的结合：损失的全长1，乳腺癌发病初期（BRCA1基因 ）在乳腺上皮细胞中的基因，肿瘤蛋白p53（TP53）种系单倍剂量不足，和乳腺上皮细胞靶向上调雌激素受体​​α（ERA）的表达在乳腺癌的发展，在100％的BRCA1基因两侧装接loxP（F）11/f11/Mouse乳腺肿瘤病毒（MMTV）-Cre/p53 + / -/tetracycline-operator（ TET-运）-ER/MMTV-reverse“四环素反式激活（rtTA 12个月的年龄在小鼠相比，比例较低报道的 BRCA1 f11/f11/MMTV-Cre/p53 + / -小鼠没有时代的过度表达（约50 - 60％）和BRCA1基因f11/f11/MMTV-Cre没有TP53 haploinsuf的小鼠效率（&lt;5％）。 癌前原发性乳腺上皮细胞的行为的动态时间推移成像揭示在细胞的行为差异，在静态的组织切片，不太容易理解。在细胞增殖和分化的改变主要观察乳腺细胞从人类BRCA1基因突变携带者。2创造的单细胞悬液，主要从正常乳腺上皮细胞和基因工程小鼠产生切除的乳腺组织通过酶的解离。时间的推移观看影像，以评估机制和时机的细胞集落的外观和细胞的形态学变化包括上皮 - 间质转化（EMT）和细胞凋亡的发生率。细胞命运的图的生成，促进细胞分裂（电池寿命），并且确定的细胞分裂的模式之间的时间的长度的定量通过使用单细胞的跟踪。蒂姆“跟踪工具（TTT）是公开可用的软件，用于生成单细胞的命运地图。其效用在阐明细胞命运的机制已经建立4,5检查正常造血干细胞发育6-9和神经元的产生。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> EpiCult-B的基础培养基鼠标</td><td>干细胞技术</td><td> 05610 </td><td></td></tr><tr><td> EpiCult-B扩散补充鼠标</td><td>干细胞技术</td><td> 05612 </td><td></td></tr><tr><td>重组人表皮生长因子（rhEGF） </td><td>干细胞技术</td><td> 02633 </td><td></td></tr><tr><td>胶原酶/透明质酸酶</td><td>干细胞技术</td><td> 07912 </td><td></td></tr><tr><td>一次性Scapels </td><td>羽毛</td><td> 2975第10 </td><td></td></tr><tr><td> Hanks&#39;平衡盐溶液</td><td>干细胞技术</td><td> 37150 </td><td></td></tr><tr><td>一mmonium氯化物</td><td>干细胞技术</td><td> 07800 </td><td></td></tr><tr><td> Tryspin-EDTA </td><td>干细胞技术</td><td> 07901 </td><td></td></tr><tr><td>中性蛋白酶</td><td>干细胞技术</td><td> 07913 </td><td></td></tr><tr><td> DNA酶I </td><td>干细胞技术</td><td> 07900 </td><td></td></tr><tr><td> 40微米的细胞过滤器</td><td>干细胞技术</td><td> 27305 </td><td></td></tr><tr><td> FBS </td><td>干细胞技术</td><td> 06100 </td><td></td></tr><tr><td> PenStrep </td><td> Gibco公司</td><td> 15140 </td><td></td></tr></tbody></table>

time-lapse imaging of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer

延时成像可以用来比较原代培养的癌前来自不同的遗传工程小鼠模型的乳腺癌的乳腺上皮细胞的行为。例如，之间的时间（电池寿命）的细胞分裂，凋亡细胞的数量，进化形态的变化，和集落形成机制，可以量化和比较携带特定遗传病变的细胞。原发性乳腺上皮细胞培养物产生的未扪及肿瘤的乳腺。有明确的分离从邻近的肌肉，仔细切除腺体，淋巴结被删除，和切碎的乳腺组织，然后用酶解和过滤所产生的丰富的乳腺上皮细胞的单细胞悬液。单细胞悬液，接种和内直接放置在显微镜下活细胞成像的孵化室。十六个650微米×700微米领域的一个4x4的配置每一个W埃尔的6孔板中的成像，每15分钟，连续5天。时间推移的影像检查，直接测量细胞的行为，可以包括细胞集落形成的机理和频率范围内的第一个24小时的电镀细胞（聚集与细胞增殖），细胞凋亡的发生率，并逐步形态的变化。单细胞跟踪是用来产生用于测量个别细胞寿命和调查的细胞分裂模式的细胞命运地图。定量数据进行统计分析，以评估与特定的遗传病变显着的行为差异。

上皮细胞和成纤维细胞，可以区分细胞的形态。上皮细胞有一个长方体形状（ 图1A-B）和形式的细胞集落（ 图1A）。成纤维细胞，基质细胞的类型，有一个细长的形态（ 图1C）。 进行了舍入细胞，并漂浮在成像（ 图2A-D）的发病。连接到该板后，他们变得平坦，并表现出立方形型外观（ 图2E中的H）。按4天的培养?...

Watch this Scientific Journal Video about Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer at JoVE.com

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

时间延时成像是用来评估初级癌前来自乳腺癌风险遗传工程小鼠模型，以确定是否有特定的行为参数之间的相关性和不同的遗传病变的乳腺上皮细胞的行为。

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Research

JoVE Journal

Medicine

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

科研

医学

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

从人类乳腺成纤维细胞癌相关的成纤维细胞的实验发电（CAFS）

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

在乳腺癌细胞雌激素调节小分子RNA的剖析

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

为获取原发性卵巢癌细胞固体样品的方法

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

注射原位乳腺癌细胞到小鼠乳腺脂肪垫来研究肿瘤的生长。

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Tracking the Mammary Architectural Features and Detecting Breast Cancer with Magnetic Resonance Diffusion Tensor Imaging

跟踪乳腺建筑特色与检测乳腺癌的磁共振扩散张量成像

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

鼠标颈淋巴结高频超声成像

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

一个器官高通量系统原发性多发性骨髓瘤细胞的药物敏感性的表征

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...