A single Drosophila embryo extract for the study of mitosis ex vivo

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.