实时成像果蝇胚胎血细胞迁移
Summary
果蝇血细胞分散在整个胚胎发育。此协议演示如何安装和图像这些用荧光标记的血细胞的胚胎的迁移。
Abstract
许多研究解决细胞迁移<em>在体外</em>方法，是生物体本身，而有关生理环境。在这里，我们提出了一个协议的安装<em>果蝇</em>胚胎及随后的荧光标记血细胞实时成像，这个有机体的胚胎巨噬细胞。利用GAL4 – UAS系统<sup> 1</sup>我们的驱动器的多种荧光标记基因编码，在血细胞的标记，按照他们在整个胚胎发育疏散的表达。以下收集所需的发展阶段的胚胎，外层的绒毛膜被删除，胚胎，然后安装在卤烃油之间的疏水性，透气膜和实时成像的玻璃盖玻片。此外，如速度和方向性更高分辨率的成像，再加上使用的F -肌动蛋白和微管可以提供更详细的信息，关于这些细胞骨架的动态的荧光记者毛候鸟参数。
Protocol
制备包含一个血细胞的特定Gal4的驱动程序（ 如SRP – Gal4 的 2）和无人机控制（ 如UAS – GFP）基因编码的下荧光记者获得适当的果蝇线。苍蝇SRP – GAL4 UAS – GMA 3或crq – GAL4 UAS – GFP 4，5纯合子是特别有用的成像目的（NB GMA的是绿色荧光蛋白融合moesin肌动蛋白结合结构域），参见下面的讨论Gal4的司机和UAS的范围内构造（布卢明顿联合中心包?…
Discussion
此过程中最重要的因素是健康的胚胎有明确标记的血细胞的选择和安装不小心损坏。一旦胚胎中的卤烃油，脱水和抗一旦安装了好几个小时可以成像。在我们的手中，我们可以形象的血细胞，三个小时，胚胎或明显的照片损害可以忽略不计的脱水，采取每三分钟我们蔡司LSM510共聚焦显微镜图像与40X目标的Z – Stack。由于血细胞是高度动态的，可能有一个空间和时间分辨率之间的权衡：如果需要一个?…
Acknowledgements
该协议已经通过我们的工作与保罗马丁和安东尼圣哈辛托实验室内和合作。我们感谢布卢明顿联合中心，以其优良的服务和继续分享飞线果蝇社会。 BS目前由BBSRC项目赠款资助。第一次世界大战是由威康信托职业发展奖学金。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Cell strainer | BD Falcon | 352350 | 70μm pores | |
| Halcarbon oil 700 | Sigma | H8898 | ||
| Lumox/Petriperm dish | Sarstedt | 96077305 |
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Tags
Live ImagingDrosophila MelanogasterEmbryonic Hemocyte MigrationsIn Vitro MethodsPhysiologically Relevant EnvironmentMounting ProtocolFluorescently Labeled HemocytesGal4-uas SystemGenetically Encoded MarkersDevelopmental DispersalEmbryo CollectionOuter Chorion RemovalHalocarbon OilHydrophobic MembraneGlass CoverslipGross Migratory ParametersSpeedDirectionalityHigher Resolution ImagingF-actinMicrotubulesCytoskeletal Components
Cite This Article
Evans, I. R., Zanet, J., Wood, W., Stramer, B. M. Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations. J. Vis. Exp. (36), e1696, doi:10.3791/1696 (2010).