A protocol for culturing Drosophila melanogaster stage 9 egg chambers for live imaging
A protocol for culturing Drosophila melanogaster stage 9 egg chambers for live imaging
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DOI：
10.1038/nprot.2007.363
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Montell, Denise J.
中科院分区：
文献类型：
--
作者：
Prasad, Mohit;Jang, Anna C-C;Montell, Denise J.
本协议描述了一种方法，用于解剖卵室从完整的果蝇女性和文化条件，允许他们的生活成像，特别强调第9阶段。这个发育阶段的特征是卵母细胞的生长和图案化，外部卵泡细胞的重排和边缘细胞的迁移。虽然在体外培养的卵室的后期发展阶段早已成为可能，直到最近阶段9卵室只能保持短期存活，没有正常发展，和边缘细胞迁移完全失败。我们已经建立了培养条件，支持整个卵室发育，包括体外边缘细胞迁移。该协议使得可以直接观察野生型和突变卵室中的分子和细胞动力学，并打开了测试药理学抑制剂和使用生物传感器的大门。整个方案需要近似24小时，而用于实时成像的卵室的准备仅需要15-20分钟。
This protocol describes a method for the dissection of egg chambers from intact Drosophila females and culture conditions that permit live imaging of them, with a particular emphasis on stage 9. This stage of development is characterized by oocyte growth and patterning, outer follicle cell rearrangement and migration of border cells. Although in vitro culture of egg chambers of later developmental stages has long been possible, until recently stage 9 egg chambers could only be kept alive for short periods, did not develop normally, and border cell migration failed entirely. We have established culture conditions that support overall egg chamber development including border cell migration in vitro. This protocol makes possible direct observation of molecular and cellular dynamics in both wild-type and mutant egg chambers, and opens the door to testing of pharmacological inhibitors and the use of biosensors. The entire protocol takes similar to 24 h while the preparation of egg chambers for live imaging requires only 15-20 min.