WPS是支持的，部分资金从美国国家卫生研究院（CA129359），为防治基金会（BCTR0706967），国防部（BC084561），和塞德曼癌症中心的Susan G. Komen，而MKW的支持从美国癌症协会的奖学金（PF-09-120-01）。这项工作提供了额外的支持，成像研究中心的案例，案例综合癌症中心（P30 CA43703），囊性纤维化中心（P30 DK027651）。

1。稳定CMV启动子驱动的Renilla荧光素酶的表达<ol><li>转染和一个稳定的无性系种群的选择是首选的方法为本海肾荧光素酶报告基因的表达。这种方法获得更加一致和统一的海肾荧光素酶的表达，随后推出的额外的辅助记者的结构（ 例如 ，萤火虫荧光素酶或荧光蛋白）。 </li><li>转染编码海肾荧光素酶的表达载体，如质粒pcDNA3.1-温湿度计或另一个质粒转染的可选择标记感兴趣的恶性细胞。 </li><li>转染后，将下一个优化的抗生素浓度的转染几天周，以便隔离的单个菌落，它们随后分离单独和传代培养。 </li><li>选择≥10个人Renilla荧光素酶表达的殖民地监测的海肾表达的程度。 </li><li&gt; Renilla荧光素酶高表达量的殖民地随后进行功能性分析，以确保他们的父母（一）病理生理特性的保留，及（ii），海肾表达式的值不偏离或随时间而改变。这些措施是绝对必要的，以避免孤立和研究克隆变种/离经叛道，并验证适当的整合到基因组的Renilla的结构。 </li><li>一旦获得稳定的转染子中分离和验证，可以删除的选择压力，并放心，海肾荧光素酶的表达将在延长的时间的长度，无论是在体外和体内的保持不变。 </li></ol> 2。诱导型启动子驱动的萤火虫荧光素酶的表达，筛选和功能验证<ol><li>成pGL4荧光素酶报告质粒转染的选择标记（ 例如 ，购买亚克隆启动子的兴趣霉素），选择稳定的海肾表达（ 例如 ，潮霉素）是不同的。 </li><li>恶性细胞转染的步骤我，随后选择一个稳定的的多克隆人口的萤火虫荧光素酶表达的细胞。由于报告基因整合到基因组中的位置可以引起错误的表达和调控的影响，我们强烈建议，而不是选择为的多克隆人口的萤火虫荧光素酶表达细胞无性系种群，以确保（i）报告基因的表达更准确地反映在亲代细胞中的内源基因，及（ii）对基因表达的平均积分效应和减少跨异构细胞种群。 </li><li>设计，以稳定地同时表达海肾和萤火虫荧光素酶的恶性细胞统称为双的生物发光记者细胞，或DBR细胞。 </li><li>使用传统的&lt;em&gt;的体外细胞为基础的荧光素酶报告基因检测，验证，DBR细胞调节萤火虫荧光素酶的表达，无论是正面或负面的，一致的，用这些载体转染的亲本细胞中观察到的方式。同样，验证CMV启动子驱动表达的海肾受各种细胞刺激或处理条件。 </li><li>这样做，文化DBR和亲代细胞在24孔板中，并与原来的CMV-海肾和启动子-萤火虫其后瞬时共转染亲代细胞构造用于生成DBR细胞。 </li><li>随后，治疗DBR和转染的亲本细胞被称为调节启动子的兴趣，随后定量使用Promega公司的双荧光素酶检测试剂盒的萤火虫和海肾发光的因素或药物制剂。 </li></ol> 3。 ，建立4T1原发性乳腺肿瘤<ol><li>转移性乳腺癌细胞，4T1我们重新发现缺乏外膜啮齿动物病原体的设计，稳定地表达了CMV启动子驱动的海肾荧光素酶（转染CMV-Renilla荧光素酶HYGRO）和SBE-驱动的萤火虫荧光素酶（pGL4.2-SBE-萤火虫荧光素酶PURO）如上所述。这些乳腺癌细胞的查询结果自发转移，主要在肺部形成原位植入。 </li><li>不应该被允许4T1细胞达到汇合在他们的传统的2 -维组织培养系统中的传播，和他们应传代他们在体内接种的前一天。 </li><li> 4T1细胞，通过胰蛋白酶消化分离，充分洗涤后，在生长培养基中，并在PBS中稀释至2×10 5个细胞/ ml的浓度，并立即在冰上储存。 </li><li>应该是带有感应剂量的3％异氟醚和剂量为1％异氟烷维持麻醉下在麻醉的雌性Balb / C小鼠（4-6周龄）。准备注射部位的擦拭，70％的异丙醇。使用钳子，轻轻抓住并提起第4腹股沟的乳垫。小心地将27.5号针头斜面的一面朝上，并直接注入乳腺脂肪垫下的乳头，特别注意不要推针进入腹腔。松开密封压盖和慢慢注入50微升的细胞悬浮液（1×10 4个细胞）到乳腺脂肪垫。 </li></ol> 4。最初的双发光成像<ol><li>后立即嫁接到乳腺脂肪垫的4T1细胞，而小鼠仍麻醉，成横向的尾静脉注入100μl的RediJect腔肠素。这是最适底物浓度的供应商和耐受性的动物。 </li><li>立即将鼠标异氟醚鼻锥内的IVIS 200成像系统，获得一个0.5分钟的荧光图像。注入和图像采集之间的效率是非常重要的，因为信号Renilla荧光素酶急剧下降约30秒后注射。将鼠标到它的笼子里，让它恢复≥1小时，以确保任何残留Renilla荧光素酶信号已经消失，鼠标已经完全从麻醉中恢复过来。 </li><li>管理通过 IP注射150毫克/公斤，D-荧光素钾盐，并等待5分钟。这是最佳的浓度，D-荧光素，它提供了一个稳定的发光信号为5-15分钟后，注入试验动物。麻醉在IVIS-200鼠标采用isoflourane和替换，照顾的动物的位置在一个非常相似的位置，相对于原来的海肾收购。整体强度本萤火虫信号的依赖的启动子的活性的利益，因此，可视化萤火虫荧光素酶的活性可能会延长收购时间。 </li><li>使用的IVIS生活图片软件设置为“光子”模式，建立有价值ES Renilla的和萤​​火虫收购的作为一种手段来建立基线的相对发光率（RLR）。 </li></ol> 5。纵向发光成像<ol><li> 4T1细胞是高度侵略性的，例如，1×10 4个细胞接种通常会导致扪肿瘤形成，1星期内，并在4-5周的动物的杀伤力。 4T1肿瘤有一种内在的倾向溃烂第4周。溃疡肿瘤的生长和维持的可能需要单独的IACUC批准。本文所示的4T1肿瘤的研究已批准的IA​​CUC在凯斯西储大学。 </li><li>如上所述，被注入小鼠应每周RediJect腔肠素监测一般肿瘤的生长和转移，以及与D-荧光素，肿瘤的发展的各个阶段期间监测路径特定的信令。小心，以确保每次Renilla荧光素酶信号已完全消退之前收购萤火虫荧光素酶信号。 </li><li>由于4T1肿瘤发展和进步，海肾荧光素酶的收购应被归一化到在作为一种手段正常化和跟踪初级肿瘤生长的肿瘤细胞接种的时间的测量值的初始海肾。更重要的是，计算RLR值随着时间的推移，将建立单独信令系统相对于肿瘤的生长和发展的时间调节。 </li><li>在3-4周后，的4T1细胞移植到乳腺脂肪垫明显肺转移明显。比较计算原发肿瘤的肺RLR值与建立个人相对于肿瘤的生长和转移的信号系统调节的空间。 </li></ol> 6。双生物发光成像的两个独特的细胞类型中的一种动物<ol><li>工程师1乳腺癌细胞系中稳定表达的CMV启动子驱动的海肾荧光素酶作为如上所述。重复此工程过程使用CMV启动子驱动的第二不同的乳腺癌细胞株上的萤火虫荧光素酶。 </li><li>在此，我们混合Renilla荧光素酶表达的4T1细胞的转移性和同源的萤火虫荧光素酶的表达4T07同行10，11。这些混合的乳腺癌细胞群的不同比例的如上述那样的乳腺脂肪垫随后注入。 </li><li>立即获得萤火虫和海肾荧光素酶的图像，到建立初始RLRs，代表一个给定的接种细胞混合物。 </li><li>纵向双生物发光成像将可视化和跟踪变化的原发肿瘤内的细胞的组合物，以及每个乳腺癌衍生物相对于肿瘤的生长和发展的时空转移的。 </li><li>可选地，个体乳腺癌细胞种群可以在小鼠接种到不同的位置（ 即左，右腹部乳腺脂肪垫）评估的系统性影响这两个群体表现出了彼此在各阶段的肿瘤生长和转移的。 </li></ol> 7。代表性的成果： 双生物发光成像的主要优势在于一个事实，即每幅图像是内部一致的控制，如持续信号来自原发肿瘤的功能作为一个重要的指标来衡量整体成功或失败的每个海肾和萤火虫收购。这方面的摄像程序的过程中是特别重要的收购海肾荧光素酶的活性，其腔肠素的基板是高度敏感的氧化，在其标注自动致发光的结果。图1A示出了该副作用的一个例子，它立即表现作为非特异性的荧光信号是来自于肠道中，不是既定的原发肿瘤。通常情况下，这些非特异性的腔肠素信号transpirË下列失败静脉注射本海肾荧光素酶底物。然而，一旦健壮原发性肿瘤衍生的海肾信号已经获得（图1B中， 左图像 ），它是安全的，继续进行在捕获来自成像萤火虫荧光素酶（图1B中， 右面板 ），其D-荧光素的特定的信令通路测量IP注射后的基板是高度稳定的，并产生背景自动发光的可以忽略不计的水平。 <img alt="图1" src="/files/ftp_upload/3245/3245fig1.jpg" /> 图1。，获取海肾和萤火虫-衍生的生物发光图像计算体内 RLRs。（A）的一个例子的一个失败的IV注射腔肠素，导致非特异性信号从肠道。圈和PT表示原发肿瘤的大致位置。（B）鼠标一个成功的海肾收购轴承4T1原发肿瘤和pulmonary转移（左图脂肪垫的植入4周后）。的相应的萤火虫的收购使计算从原发肿瘤和肺转移的RLRs。（C）原发肿瘤和肺转移RLRs的图形表示从小鼠B组（n = 4）计算的。

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没有利益冲突的声明。

生物发光成像技术的绝对权力在于他们有能力进行量化复杂的纵向研究，这里涉及使用积极的4T1乳腺癌细胞的肿瘤生长和转移的。因为这些程序依赖于两种荧光素酶报告基因构建体的稳定整合，这些技术可以很容易地适应和翻译成其它癌细胞系中的不同肿瘤的潜伏期和转移能力。这里所示的结果相似，算出具体RLRs内缓慢发展的原发性肿瘤和其最终转移允许实时时空识别特定的信令转移性肿瘤进展的，不论它们的持续时间的延迟期间蒸发的事件。经鉴定特异性启动子调控事件，重要的是切除原发肿瘤和转移病灶的性能标准免疫组化和差异升的基因表达分析，以验证的内源基因和/或蛋白质的类似的调节。 从技术上讲，双生物发光分析的主要挑战在于在相对短的时间Renilla荧光素酶信号。因此，这种成像技术需要显着优化的尾静脉注射程序，以及单独注射的动物的直接成像，一个过程是耗费时间和成像的萤火虫荧光素酶的相对有点低效。最近，Promega公司推出“Viviren”，这表示第二代的荧光素酶的基板，其站点通过酯化氧化被阻塞，直到的这种分子增益进入细胞，在该点的分子是迅速脱酯化。总的来说，这种新颖的基板有效地降低自动发光和非特异性的修改和/或耦合到海肾诱导的自动发光12的退化。在这样做时，这种新的renill荧光素酶底物产生更明亮的发光信号，但过高的成本与获取和使用“Viviren”提供必要的研究相对较少，在双生物发光分析，以评估该基板上的整体效用。最后，任何生物发光测定法的灵敏度是关键取决于操作性中获取单个光单元的CCD照相机。事实上，这些技术的提高，我们预计在未来我们的能力，可视化复杂的光介导的生物发光反应，可能会蒸发，有效地自由移动的动物13点。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td> pGL4.2 PURO [luc2 / PURO]向量</td><td> Promega公司</td><td> E6751 </td></tr><tr><td> Glomax的多个-idection系统</td><td> Promega公司</td><td></td></tr><tr><td> IVIS 200系列</td><td>卡尺生命科学</td><td></td></tr><tr><td>双荧光素酶报告基因检测系统</td><td> Promega公司</td><td> E1960 </td></tr><tr><td> Rediject腔肠素-H </td><td>卡尺生命科学</td><td> 760506 </td></tr><tr><td> D-荧光素K-盐</td><td>卡尺生命科学</td><td> 122796 </td></tr></tbody></table>

in vivo dual substrate bioluminescent imaging

我们的理解和乳腺癌细胞从原发肿瘤转移部位过境时增加了一个特殊的速度问世以来， 在体内生物发光成像技术1-3。事实上，能够在一个单一的队列的动物，而不是牺牲个人组动物在特定的试验时间，已经彻底改变了研究人员研究乳腺癌转移的研究完成的纵向定位和量化肿瘤的生​​长。不幸的是，目前的方法排除蒸发乳腺癌细胞（I）内原发肿瘤发展，（二）传播的整个身体，及（iii）重新开始增生性节目的网站在细胞信号传导系统发生重大变化，实时评估转移性病变。然而，最近的进步，生物发光成像现在使人们有可能同时量化特定spatiotempor人的基因表达的变化作为一个功能通过使用双衬底发光反应的肿瘤的发展和转移进展。要做到这一点，研究人员利用从萤火虫（Photinus pyralis）和海三色堇（ 海肾肾形肾状 ），这两种反应以前促进了它们的广泛使用在体外细胞的相互排斥的基板分离的两个光产生的荧光素酶的酶基于报告基因检测4。在这里，我们证明了这两种酶，使得一个发光反应具体的显影的肿瘤的大小和位置的标记，而所述第二发光反应作为一种手段来可视化特定的信令系统的激活状态，在不同的阶段，肿瘤在体内效用和转移的发展。因此，本研究的目标是2倍。首先，我们将介绍必要的步骤来构建双生物发光记者细胞系，以及那些需要，以便使用在特定的步骤转移级联的可视化的基因表达调控的时空。使用4T1乳癌转移模型中，我们表明Smad蛋白绑定元素的合成（SBE） 的体内活性的启动子急剧下降相比，在原发肿瘤4-6测量肺转移。最近，被证明是调控乳腺癌转移在原发肿瘤微环境和反应性间质的变化，包括那些发生在成纤维细胞和免疫细胞浸润7-9。因此，我们的第二个目标将是证明在监测的增长和本地化两个独特的细胞群窝藏在一个单一的动物在乳腺癌的生长和转移的的双生物发光技术的效用。

Watch this Scientific Journal Video about In vivo Dual Substrate Bioluminescent Imaging at JoVE.com

In vivo Dual Substrate Bioluminescent Imaging

在这里，我们描述的方法建设，可视化和量化的萤火虫和Renilla荧光素酶表达的转移性乳腺癌细胞的生长和转移的生物发光反应<em在体内</em&gt;。

在体内的双基体生物光学成像

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

in-vivo-dual-substrate-bioluminescent-imaging

Research

JoVE Journal

Medicine

In vivo Dual Substrate Bioluminescent Imaging

15.4K 次观看.  Case Western Reserve University. 在这里，我们描述的方法建设，可视化和量化的萤火虫和Renilla荧光素酶表达的转移性乳腺癌细胞的生长和转移的生物发光反应

视频: In vivo Dual Substrate Bioluminescent Imaging

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

发光法小鼠肝肿瘤模型

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

科研

医学

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.

A dual-mode imaging system was developed for non-contact assessment of cutaneous tissue oxygenation and vascular function.

双模式成像，皮肤组织氧合和血管功能

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health ...

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

随后的三维颅内植入在体内</em生物发光成像鼠脑胶质瘤

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

立体定向颅内植入和<em在体内</em生物发光成像的肿瘤异种移植的小鼠模型系统的多形性胶质母细胞瘤

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

在肿瘤血管生成中使用荧光共聚焦电视显微镜活体成像

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

生物荧光原位模型胰腺癌进展

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

在体内 19女性 MRI对细胞跟踪

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the pathophysiological role of neuroplasticity in GI disorders remains unknown. Novel experimental models which allow simulation and modulation of GI neuroplasticity may enable enhanced appreciation of the contribution of neuroplasticity in particular GI diseases such as pancreatic cancer (PCa) and chronic pancreatitis (CP). Here, we present a protocol for simulation of pancreatic neuroplasticity under in vitro conditions using newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons. This dual-neuron approach not only permits monitoring of both organ-intrinsic and -extrinsic neuroplasticity, but also represents a valuable tool to assess neuronal and glial morphology and electrophysiology. Moreover, it allows functional modulation of supplied microenvironmental contents for studying their impact on neuroplasticity. Once established, the present neuroplasticity assay bears the potential of being applicable to the study of neuroplasticity in any GI organ.

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuronal plasticity is an increasingly recognized, but insufficiently understood feature of the gastrointestinal (GI) tract. Here, in the example of human pancreatic disorders, we present an in vitro neuroplasticity assay for the study of neuronal plasticity in the GI tract at both morphological and functional level.

模拟胰腺神经可塑性：<em&gt;体外</em&gt;双神经元可塑性分析

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

顺序<em&gt;在体内</em&gt;成骨干/祖细胞在骨折修复的影像学

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

化疗引起的血管毒性 - 实时<em&gt;在体内</em&gt;船舶减值成像

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...