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Nat Protoc
2013 Feb 01;82:310-24. doi: 10.1038/nprot.2013.003.
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A single Drosophila embryo extract for the study of mitosis ex vivo.
Telley IA, Gáspár I, Ephrussi A, Surrey T.
???displayArticle.abstract??? Spindle assembly and chromosome segregation rely on a complex interplay of biochemical and mechanical processes. Analysis of this interplay requires precise manipulation of endogenous cellular components and high-resolution visualization. Here we provide a protocol for generating an extract from individual Drosophila syncytial embryos that supports repeated mitotic nuclear divisions with native characteristics. In contrast to the large-scale, metaphase-arrested Xenopus egg extract system, this assay enables the serial generation of extracts from single embryos of a genetically tractable organism, and each extract contains dozens of autonomously dividing nuclei that can be prepared and imaged within 60-90 min after embryo collection. We describe the microscopy setup and micropipette production that facilitate single-embryo manipulation, the preparation of embryos and the steps for making functional extracts that allow time-lapse microscopy of mitotic divisions ex vivo. The assay enables a unique combination of genetic, biochemical, optical and mechanical manipulations of the mitotic machinery.
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