This research is supported by funding from CBCF (to ZW).

1.准备实时成像单元格<ol><li> 细胞培养和播种 <ol><li>使用选择用于抗多西他赛（MCF-7 的TXT）和它们的非抗性亲代细胞系（MCF-7，CC）MCF-7乳腺癌细胞。详细的选择过程并且这些选择的细胞系的表征先前24描述。 </li><li>生长于10cm培养皿中的所有细胞在37℃下在由90％的Dulbecco改进的Eagle培养基（DMEM）和胎牛血清（FBS）的10％，并补充有非必需氨基酸介质。保持在5％ 的 CO 2气氛中的介质。保持在5nM的多西他赛的MCF-7 的TXT细胞。 </li><li>种子细胞盖玻片上的实时成像。通过用70％乙醇漂洗它们，然后将它们暴露于紫外线过夜消毒24毫米涂覆聚-L-赖氨酸盖玻片。放置盖玻片成35mm的6孔培养板上。 PL在每口井癿盖玻片。 </li><li>除去从10厘米的培养皿的培养基，并用PBS洗涤细胞。添加0.5毫升胰蛋白酶-EDTA的的菜分离从盘的细胞。添加5毫升培养基的培养皿中以中和胰蛋白酶的细胞的分离如下。 </li><li>计数细胞用血细胞计数器确定每单位体积电池的数目。 </li><li>加约10 5个细胞每孔的基础上每单位体积的细胞数量。轻轻摇动后，将板放回孵化器，以使细胞附着在盖玻片。 </li></ol></li><li> GFP标记α微管蛋白的表达 <ol><li>为了测定微管动力学，转导与GFP标记α微管蛋白的细胞根据制造商的协议商业GFP转导控制（ 例如，BacMam）。 </li><li>培养在井中的细胞36小时在37℃的细胞培养孵化器，以允许玉米完整粘附和60％汇合。 </li><li>计算GFP标记α微管蛋白的适当体积添加到各孔中，根据以下公式: <img alt="方程" src="/files/ftp_upload/55027/55027eq1.jpg" /> 细胞的数量是细胞在井在标签的时间的估计的总数目和所需的PPC是每个细胞的粒子数。 注:播种在MCF-7细胞应该一倍，从10 5至2×10 5细胞数为20的期望的PPC，需要为每个盖玻片的体积因此是40微升。对于不同的小区，体积算出基于上述公式可能不是最好的。确切的量应根据GFP微管蛋白的实际表达水平进行调整。 </li><li>通过倒置混合GFP标记α微管蛋白几次，以确保均匀的溶液。不要旋涡。 注:对于控制中，空（控制）试剂没有任何哺乳动物的基因元件和可用于帮助确定潜在杆状病毒介导的作用和背景荧光。非针对性的GFP转导的控制也可使用，这将照亮整个细胞。 </li><li>替换用新鲜培养基的培养基中。添加2mL的新培养基中，40微升GFP标记α微管蛋白的每个孔中。 </li><li>轻轻摇动板以允许与所述培养基GFP标记α微管蛋白的完全混合。返回板到培养培养箱孵育24小时，以允许GFP微管蛋白的表达。 </li></ol></li><li> 处理细胞多西他赛 注:此实验是测试多西他赛的微管动态不稳定性的影响。因此，该细胞与多西紫杉醇的所需浓度进行处理。 <ol><li>制备的培养基由90％的DMEM和10％FBS的和补充有非必需氨基酸。使用DMEM培养基无酚红，因为酚红可以与fluorescen干扰GFP微管蛋白的CE。添加多西他赛（范围从10nM至10μM）的所需的浓度。多西紫杉醇的储备溶液是在二甲亚砜（DMSO）的10mM。 </li><li>预暖在组织培养培养箱含多西紫杉醇的培养基至37℃。 </li><li>替换与含多西紫杉醇的培养基正常培养基并孵育在组织培养箱30分钟。应该有每孔至少为3的盖玻片为每个多西紫杉醇的浓度。 </li></ol></li></ol> 2.实时成像，以检查微管动态失稳<ol><li> 设置系统 <ol><li>在保持在37℃和5％的CO 2的腔室进行实验。由时间流逝用装有60X，在一个倒置荧光显微镜和CCD照相机1.42 NA油物镜的显微镜系统获取荧光图像。 </li><li>设置了实时成像的细胞。预暖两个样本保持器和在中至37℃。选择一个盖玻片检查。安装在试样保持器的兴趣盖玻片并用1mL在步骤1.3.1制备的含多西紫杉醇的培养基孵育它。 </li></ol></li><li> 微管动态不稳定的实时成像 <ol><li>确定将被用于成像的细胞。他们应该是平的，有表达GFP微管蛋白的荧光强度高，并有明确的微管结构。 </li><li>选择组从步骤2.2.1确定细胞的一个单元首次检验。集中在识别的小区的周边区域。设置了曝光时间的程序和图像采集的频率。由于曝光时间一般是0.5秒，图像将每两个s收购。 </li><li>允许步骤，一个额外的20分钟从1.33至2.2.3。因此，恰好在加入多西他赛之后50分钟，如在步骤1.3.3所述，开始成像（这是标准化）。 </li><li>记录微管ðynamics 2分钟，拍摄照片，每2秒。总共获得用于识别的小区60的图像。 </li><li>移动到下一个识别的小区，并重复步骤2.2.2至2.2.4。重复上述步骤，直到5细胞已被成像。当务之急是做到这一点的时间达到加成多西他赛之后70分钟，然后，如在步骤1.3.3说明。 </li><li>对于每个处理条件，重复步骤2.2.1至2.2.5 3种不同的盖玻片。总体而言，图像15细胞，这是足够的数据来衡量的微管动态不稳定。 </li></ol></li></ol> 3.图像处理和数据分析<ol><li> 图像反卷积 <ol><li>去卷积所获取的图像，以实现高品质。打开装有显微镜系统去卷积程序。 </li><li>单击"进程"，然后选择"反卷积"。图像文件拖动到"输入"窗口，点击"做。"该图像文件将被去卷积，并且反褶积文件将被自动保存为一个新文件。 </li></ol></li><li> 视频生成 <ol><li>点击反褶积图像文件与配套软件打开。 </li><li>点击"文件"，选择"另存为电影"。选择"电影格式"为"AV"和"动画风格"为"前进"。将"压缩质量"到"100％"和"帧速率"到"5张/秒。" </li><li>检查"通过时间动画"，并单击框"做吧。"视频将产生。 </li></ol></li><li> 微管缩短和增长速度的测量 <ol><li>确定将被用于测量的微管。对于每个小区，只有少数微管就能有其缩短或增长遵循清晰，完整。 </li><li>检查视频的每个帧，以确定显示微管一个的起始长度的帧第二最延长或缩短微管的帧。测量两个微管和两帧之间所经过的时间之间的长度变化。 </li><li>通过时间除以长度变化计算增长或缩短的速度（单位是微米/秒）。对于每个处理条件，遵循至少10个细胞，并测量至少20微管。 </li><li>计算每个处理条件的平均值和标准误差。分析使用Student测试条件之间的统计差异。报告在表和/或图表中的数据。 </li></ol></li></ol>

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作者宣称，他们没有竞争的经济利益。

有两种主要的方法来测量微管动态不稳定性: 在体外和体内。在体外方法中，纯化的微管蛋白是用来测量与计算机增强的时间推移微分干涉反差显微镜微管动态不稳定性。在体内方法中，显微注射荧光微管蛋白，或表达GFP的微管蛋白，掺入微管。微管的动力学（生长和缩短）然后通过时间推移在间期细胞10，20，25</su...

<html>乳腺癌死亡率的主要原因就是通过转移1，2。紫杉烷类，如多西他赛和紫杉醇，目前用作一线转移性乳腺癌2，3，4，5，6的治疗方案。他们是一群扰乱微管动力学微管靶向代理（MTA）的一部分。然而，在治疗性治疗使用紫杉烷类的最大挑战之一是在癌细胞中紫杉烷抗性，从而导致疾病复发7的发展。耐药占转移性乳腺癌7之间所有死亡的90％以上。 微管是由α-和β微管蛋白异源二聚体的聚合形成类="外部参照"&gt; 8，9。微管动力学的精确调节是许多细胞功能，包括细胞极化，细胞周期进程，细胞内运输和细胞信号传导的重要。微管和它们的动态的失调会扰乱细胞的功能，导致细胞死亡10,11。取决于它们怎样导致此失调，MTA药物可归类为微管稳定剂（即紫杉烷）或微管destabalizing剂（ 即，长春花生物碱或秋水仙碱点结合剂）20。尽管微管的质量的相反的效果，以足够的剂量，这两个类可以通过其对微管动力学21作用杀死癌细胞。 紫杉烷类通过稳定微管主轴12主要作用，导致染色体错位。在纺锤体装配检验点（SAC）的后续永久激活逮捕的有丝分裂细胞。延长的有丝分裂停滞然后导致凋亡13,14。紫杉烷与通过对β微管蛋白8，15，这是只有在组装的微管蛋白16存在的紫杉烷的结合位点的微管相互作用。 对紫杉烷抗性多种机制已经提出9,17。这些机制包括由于药物外排蛋白与紫杉类药物特异性抵抗力5，9，18，19的表达了一般多药耐药性。例如，耐紫杉类癌细胞可能已经改变了某些β桶的表达和功能同型球蛋白5，9，19，20，21，22，23。通过使用体内方法测量微管动态不稳定，我们表明，相对于非抗性，父母的MCF-7 的CC单元17时，耐多西紫杉醇的MCF-7 的TXT细胞的微管动力学是不敏感的多西他赛治疗。 为了更好地了解的MTA的功能，并在癌细胞紫杉烷抗性的确切机制，重要的是测量微管动力学。这里，我们报告这样的体内方法。通过使用实时成像结合GFP标记的微管蛋白在细胞中的表达，我们可以测量的MCF-7 的TXT和MCF-7 的CC细胞和Wi的微管动力学thout多西紫杉醇治疗。研究结果可以帮助我们设计出能够克服阻力紫杉更加有效的药物。

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Sigma-Aldrich</td><td>D5796</td><td></td></tr><tr><td>Non-essential amino acids</td><td>Life Technologies, Invitrogen</td><td>11140-050</td><td></td></tr><tr><td>FBS</td><td>Gibco, Invitrogen</td><td>12483</td><td></td></tr><tr><td>Anti-Anti (100x)</td><td>Life Technologies, Invitrogen</td><td>15240-062</td><td></td></tr><tr><td>docetaxel</td><td>Sigma-Aldrich</td><td>01885-5mg-F</td><td></td></tr><tr><td>DMEM phenol red-free</td><td>Gibco, Invitrogen</td><td>21063</td><td></td></tr><tr><td>CellLight Reagent *BacMam 2.0* GFP-tubulin</td><td>ThermoFisher Scientific</td><td>C10613</td><td>Key reagent for expressing GFP tubulin in cells</td></tr><tr><td>CellLight Reagent *BacMam 2.0* GFP</td><td>ThermoFisher Scientific</td><td>B10383</td><td>Control</td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma-Aldrich+B9:AA9</td><td>472301</td><td>for dissoving decetaxel</td></tr><tr><td>22-mm glass coveslip</td><td>Fisher Scientifics</td><td>12-545-101</td><td></td></tr><tr><td>6-well culture plate</td><td>Greiner Bio-One International</td><td>6 Well Celi Culture Plate</td><td></td></tr><tr><td>DeltaVision Microscopy Imaging Systems</td><td>GE Health</td><td></td><td>This system is equipped with weather station for controlling temperature and CO2. It also equipped with Worx Software for deconvolution and time lapse control.</td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>ThermoFisher Scientific</td><td>25200056</td><td></td></tr><tr><td>Bright-Line Hemacytometer Set, Hausser Scientific</td><td>Hausser Scientific, Distributed by VWR</td><td>Supplier No.: 1492 VWR No.:15170-172</td><td></td></tr></tbody></table>

live imaging to study microtubule dynamic instability in taxane-resistant breast cancers

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane resistance in cancerous cells drastically reduces the effectiveness of the drugs' long-term usage. Accumulated evidence suggests that the mechanisms underlying taxane resistance include both general mechanisms, such as the development of multidrug resistance due to the overexpression of drug-efflux proteins, and taxane-specific mechanisms, such as those that involve microtubule dynamics. 
Because taxanes target cell microtubules, measuring microtubule dynamic instability is an important step in determining the mechanisms of taxane resistance and provides insight into how to overcome this resistance. In the experiment, an in vivo method was used to measure microtubule dynamic instability. GFP-tagged &#945;-tubulin was expressed and incorporated into microtubules in MCF-7 cells, allowing for the recording of the microtubule dynamics by time lapse using a sensitive camera. The results showed that, as opposed to the non-resistant parental MCF-7CC cells, the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitive to docetaxel treatment, which causes the resistance to docetaxel-induced mitotic arrest and apoptosis. This paper will outline this in vivo method of measuring microtubule dynamic instability.

用这里介绍的协议中，我们研究了在正常（MCF-7，CC）和耐多西紫杉醇（MCF-7 TXT）乳腺癌细胞的微管动力学的多西紫杉醇的效果。两组图像显示在微管的生长和缩短多西他赛（0.5μM）的在MCF-7 的CC和MCF-7 的TXT细胞（ 图1A）的影响。 我们还计算微管生长或缩短的速率在这些条件对两种?...

Watch this Scientific Journal Video about Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers at JoVE.com

Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells.

实时成像来研究抗紫杉烷乳腺癌微管动态失稳

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane ...

live-imaging-to-study-microtubule-dynamic-instability-taxane

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The Cytoskeleton II: Microtubules and Intermediate Filaments

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7.5K 次观看.  University of Alberta. In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells. 

视频: Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the beginning and end of an experiment using a phase-contrast microscope, and the migration abilities of cells are evaluated by the closure of wounds. However, cell movement is a dynamic phenomenon, and a conventional method does not allow for tracking single-cell movement. To improve current wound-healing assays, we use live-cell imaging techniques to monitor cell migration in real time. This method allows us to determine the cell migration rate based on a cell tracking system and provides a clearer distinction between cell migration and cell proliferation. Here, we demonstrate the use of live-cell imaging in wound-healing assays to study the different migration abilities of breast epithelial cells influenced by the presence of TIP60. As cell motility is highly dynamic, our method provides more insights into the processes of wound healing than a snapshot of wound closure taken with the traditional imaging techniques used for wound-healing assays.

Here, we present the real-time monitoring of cell migration in a wound-healing assay using TIP60-depleted MCF10A breast epithelial cells. The implementation of live-cell imaging techniques in our protocol allows us to analyze and visualize single-cell movement in real time and across time.

TIP60 短暂耗竭后乳腺上皮细胞迁移的活体影像学研究

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the ...

科研

癌症研究

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

生物发光和微型CT成像监测肿瘤转移和溶骨性病变

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

医学

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

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

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

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis 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 ...

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

长期活细胞成像对紫杉醇反应细胞命运的评估

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

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

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

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 ...

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

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

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 ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

在髓系细胞动态实时成像<em&gt;装甲运兵车<sup&gt;最小/ +</sup</em&gt;肠道肿瘤通过旋转盘共聚焦显微镜

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

免疫与感染

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked&#160;bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

通过TIRF显微镜 在体外 同时可视化交联和单微管的动力学

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...

生物化学

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

透过玻璃看:时间推移显微镜和单细胞的纵向跟踪研究抗癌治疗

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

实时多细胞动力学延长时间推移活体成像在肿瘤微环境

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

实时成像来研究抗紫杉烷乳腺癌微管动态失稳

摘要

探索更多视频

此视频中的章节

TIP60 短暂耗竭后乳腺上皮细胞迁移的活体影像学研究

生物发光和微型CT成像监测肿瘤转移和溶骨性病变

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

长期活细胞成像对紫杉醇反应细胞命运的评估

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

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

在髓系细胞动态实时成像

通过TIRF显微镜在体外同时可视化交联和单微管的动力学

透过玻璃看:时间推移显微镜和单细胞的纵向跟踪研究抗癌治疗

实时多细胞动力学延长时间推移活体成像在肿瘤微环境

实时成像来研究抗紫杉烷乳腺癌微管动态失稳

摘要

探索更多视频

此视频中的章节

TIP60 短暂耗竭后乳腺上皮细胞迁移的活体影像学研究

生物发光和微型CT成像监测肿瘤转移和溶骨性病变

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

长期活细胞成像对紫杉醇反应细胞命运的评估

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

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

在髓系细胞动态实时成像

通过TIRF显微镜 在体外 同时可视化交联和单微管的动力学

透过玻璃看:时间推移显微镜和单细胞的纵向跟踪研究抗癌治疗

实时多细胞动力学延长时间推移活体成像在肿瘤微环境

通过TIRF显微镜在体外同时可视化交联和单微管的动力学