Name: ex vivo method for high resolution imaging of cilia motility in rodent airway epithelia
Uploaded: 2013-08-08T13:00:00
Description: Watch this Scientific Journal Video about Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia at JoVE.com

该项目是由NIH授予U01HL098180从支持国家心脏，肺和血液研究所。内容完全是作者的责任，并不一定代表官方意见，国家心脏，肺和血液研究所或国立卫生研究院

1。试剂设置 1.1解剖和成像介质 Leibovitz的L-15培养基（L15）补充有FBS（10％）和青霉素 - 链霉素（100单位/ ml青霉素G钠和100微克/毫升硫酸链霉素）气管样品收获和成像过程中使用的。 2。浅寨文化商会大会 图1所示的腔室用来装气管组织。室的地板是玻璃见底35毫米培养皿。通过为0.3mm厚的硅薄片的玻璃盘上?...

<ol>
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J. 34, 401-404 (2009).</li><li>Drummond, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+cilia+in+zebrafish.">Studying cilia in zebrafish.</a> Methods Cell Biol. 93 (08), 197-217 (2009).</li><li>Meijering, E., Dzyubachyk, O., Smal, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+cell+and+particle+tracking.">Methods for cell and particle tracking.</a> Methods Enzymol. 504, 183-200 (2012).</li><li>Sisson, J. H., Stoner, J. A., Ammons, B. A., Wyatt, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All-digital+image+capture+and+whole-field+analysis+of+ciliary+beat+frequency.">All-digital image capture and whole-field analysis of ciliary beat frequency.</a> J. 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F., Cosson, J., Huitorel, P., Blaive, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+effect+on+the+ciliary+beat+frequency+of+human+nasal+and+tracheal+ciliated+cells.">Temperature effect on the ciliary beat frequency of human nasal and tracheal ciliated cells.</a> Biology of the Cell / under the auspices of the European Cell Biology Organization. 76, 335-338 (1992).</li></ol>

测量纤毛摆动频率（CBF）是比较容易的使用高倍显微镜的目标和快速的图像采集硬件13,15，并解释了为什么脑血流量测量形成的基础研究在健康和疾病中的黏液纤毛清除。然而，当脑血流量测量是必不可少的了解黏液纤毛清除，单独测量CBF睫状肌产生的流量和纤毛摆动波形，这两者都是比较难以衡量，往往被忽视的重要性，忽略了潜在的，可能没有相关的脑血流量。例如如何增加脑血流量...

阐明遗传和环境因素会影响粘液纤毛清除功能和肺的健康1，气道上皮细胞的运动纤毛功能分析实验是重要的。成像的小鼠气道上皮细胞的简单的协议，开发提供了一种有效的方法来审问呼吸道纤毛运动的突变体和基因敲除小鼠模型，并在小鼠气管组织解剖和体外成像与高清晰度电视显微镜，呼吸道纤毛蠕动只需要基本技能。此协议在建立和完善了大规模的鼠标诱变屏幕，运动纤毛功能允许快速评估（纤毛摆动频率，纤毛摆动形状，纤毛产生的流量）患有先天性心脏疾病的伴有内脏异位2-5的突变体。 目前的技 ​​术，用于研究呼吸道纤毛蠕动，可以分组为急性体外类型或离子蒙古包长期在体外实验方法。急性实验包括人/鼻气道活检刷6,7 体外可视化和分析简单的横向气道第8。 在体外细胞培养方法利用各种技术，以产生分化的纤毛上皮细胞，如空气中的液体界面的文化或气悬浮培养9-11张。然而，这些气道上皮reciliation的技术要求非常显著的投资在时间和培训之前任何可用的纤毛上皮细胞的实验（4-6周9,10）。虽然通常用于人体临床研究急性呼吸道上皮刷活检体外分析，这种方法是不是可以使用小鼠的研究，由于加剧了机械组织损伤12。 在这个协议中所述的技术分析的谅解备忘录ê气管的气道上皮细胞不仅是简单的执行，但它不需要特殊的解剖技能，也没有任何专门的设备除了那些电视显微镜成像标准。这个简单的协议有许多优点。首先，小鼠气管组织收获是快速和容易执行，它允许大量的小鼠气道纤毛功能快速评估。这可以包括急性分析不同在体外治疗的短期效果。其次，作为体外技术，气道纤毛上皮细胞仍然连接到相关的支持组织和，从而保留相关的细胞信号通路。因此，在比较， 在体外 reciliated的气道上皮细胞，该制剂在体内的组织环境是一个更好的代表性的自然。第三，该协议允许收购了一些不同的量化参数，可以提供客观的评估运动纤毛f油膏。最后，在对比其他流动呼吸道纤毛可视化的方法，这个协议允许的纤毛纤毛摆动方向直角可视化，允许纵断面图中的纤毛，是最佳的高分辨率成像的纤毛摆动和metachronal的波代。 这个协议可以修改了一些方法来解决了广泛的实验需求等作用的药物制剂，遗传因素，环境暴露，和/或机械因素，如粘液负载上呼吸道纤毛功能和生成/维护呼吸道纤毛摆动和波传播metachronal。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Leibovitz's L-15 Medium </td>
 <td>Invitrogen </td>
 <td>21083-027 </td>
 <td>No phenol red </td>
 </tr>
 <tr>
 <td>Fetal bovine serum </td>
 <td>Hyclone </td>
 <td>SH30088.03 </td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin </td>
 <td>Invitrogen </td>
 <td>15140-122 </td>
 <td></td>
 </tr>
 <tr>
 <td>2x fine forceps </td>
 <td>Roboz </td>
 <td>RS-4976 </td>
 <td></td>
 </tr>
 <tr>
 <td>Dissection scissors </td>
 <td>Roboz </td>
 <td>RS-5676 </td>
 <td></td>
 </tr>
 <tr>
 <td>Micro dissection scissors </td>
 <td>Roboz </td>
 <td>RS-5620 </td>
 <td></td>
 </tr>
 <tr>
 <td>Scalpel </td>
 <td>Roboz </td>
 <td>RS-9801-15 </td>
 <td></td>
 </tr>
 <tr>
 <td>P1000 pipetman </td>
 <td>Gilson, Inc </td>
 <td>F123602 </td>
 <td></td>
 </tr>
 <tr>
 <td>P1000 tips </td>
 <td>Molecular BioProducts </td>
 <td>2079E </td>
 <td></td>
 </tr>
 <tr>
 <td>18 mm round glass cover slips </td>
 <td>Fisher Scientific </td>
 <td>430588 </td>
 <td></td>
 </tr>
 <tr>
 <td>Plastic 35 mm culture dishes </td>
 <td>Corning </td>
 <td>430588 </td>
 <td></td>
 </tr>
 <tr>
 <td>Glass bottom 35 mm culture dishes </td>
 <td>Warner Instruments </td>
 <td>W3 64-0758 </td>
 <td></td>
 </tr>
 <tr>
 <td>Silicone sheet 0.012" (0.3 mm) thick </td>
 <td>AAA Acme Rubber Co </td>
 <td>CASS-.012X36-63908 </td>
 <td></td>
 </tr>
 <tr>
 <td>0.20 &mu;m diameter Fluoresbrite YG Carboxylate Microspheres </td>
 <td>Polysciences </td>
 <td>09834-10 </td>
 <td></td>
 </tr>
 <tr>
 <td>Inverted microscope, with 100x oil objective and DIC filters </td>
 <td>Lecia</td>
 <td>DMIRE2 </td>
 <td>Brand is not critical. </td>
 </tr>
 <tr>
 <td>100-watt mercury lamp, epifluorescent FITC excitation/emission filters</td>
 <td>Lecia </td>
 <td></td>
 <td>Brand is not critical.</td>
 </tr>
 <tr>
 <td>Microscope stage Incubator</td>
 <td>Lecia </td>
 <td>11521749 </td>
 <td>Not required if imaging cilia at room temperature</td>
 </tr>
 <tr>
 <td>High-speed camera bright field </td>
 <td>Vision Research </td>
 <td>Phantom v4.2 </td>
 <td>Brand is not critical. Must be faster than 125 fps</td>
 </tr>
 <tr>
 <td>High-speed fluorescent camera </td>
 <td>Hamamatsu </td>
 <td>C9100-12 </td>
 <td>Brand is not critical. Must be faster than 10 fps</td>
 </tr>
 <tr>
 <td>Movie analysis software</td>
 <td>National Institutes of Health</td>
 <td>ImageJ with MtrackJ plugin</td>
 <td></td>
 </tr>
 </tbody>
</table>

ex vivo method for high resolution imaging of cilia motility in rodent airway epithelia

体外小鼠气道上皮细胞成像技术定量分析洞察黏液纤毛清除功能的重要运动纤毛功能已经确立。刚收获的小鼠气管，通过纵向切开气管肌肉和安装在浅壁室玻璃底的菜。其长轴沿气管样品趁气管肌肉纵向卷曲。这使得成像的纤毛运动，沿着整个气管长度在纵断面图。使用微分干涉显微镜和一个高速的数码相机得到允许纤毛摆动频率的定量分析和睫状波形画在200帧/秒。在成像过程中添加的荧光珠，纤毛产生的流体流动也可以被确定。协议时间跨越约30分钟，5分钟5-10分钟的样品制备室安装，和10-15分钟电视显微镜。

控制呼吸道纤毛应清晰可见，看到击败以协调的方式（ 补充电影1，电影播放放缓至15％的实际时间），纤毛摆动的方向（ 补充电影2，电影播放100％有明显的流实时）。定量的纤毛电影应该产生类似的结果如图3所示。未能收集高速DIC电影使纤毛摆动频率的定量分析（ 图3C），纤毛摆动形状的定性评估，并给出了良好的图象，而荧光珠的跟踪允许?...

Watch this Scientific Journal Video about Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia at JoVE.com

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

一个简单而可靠的可视化和量化技术，气道纤毛的蠕动和纤毛产生流量使用小鼠气管。这种技术可以被修改，以确定如何广泛的因素，包括药物制剂，遗传因素，环境暴露，和/或机械因素，如粘液负载影响纤毛运动。

在啮齿类动物呼吸道上皮纤毛运动功能的高分辨率成像的体外方法

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Research

JoVE Journal

Biology

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

从海兔的神经细胞培养的高分辨率成像生长锥

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

科研

生物学

Method for Culture of Early Chick Embryos ex vivo (New Culture)

Method for Culture of Early Chick Embryos ex vivo New Culture

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

高分辨率视频跟踪移动的，在成年果蝇。

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

在完整的果蝇幼虫体内的亚细胞分辨率成像

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

一种新型的<em&gt;体外</em小鼠胚胎心脏的培养方法

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

运动性的时空制图<em&gt;离体</em肠子&gt;准备工作

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, ...

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

高分辨率法监测IRF3的磷酸化依赖性激活

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

高分辨率成像和芽殖酵母个人星体微管动力学分析

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

Ex Vivo Method for Assessing the Mouse Reproductive Tract Spontaneous Motility and a MATLAB-based Uterus Motion Tracking Algorithm for Data Analysis

用于评估小鼠生殖道自发性的方法和用于数据分析的基于 MATLAB 的 Uterus 运动跟踪算法

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. ...