In-vivo centrifugation of Drosophila embryos.
In-vivo centrifugation of Drosophila embryos.
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DOI：
10.3791/2005
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发表时间：
2010-06-23
期刊：
影响因子：
--
通讯作者：
Welte, Michael A
中科院分区：
文献类型：
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作者：
Tran, Susan L;Welte, Michael A
纯化和分离不同类型的细胞内细胞器的主要策略是基于浮力密度的差异将它们彼此分离。然而，当细胞在离心之前被破坏时，在这种非天然环境中的蛋白质和细胞器通常不适当地彼此粘附。在这里，我们描述了一种方法来分离细胞器的密度在完整的，活的果蝇胚胎。细胞化之前的早期胚胎从群体笼中收获，并通过用50%漂白剂处理除去它们的外壳。然后将胚转移到小琼脂板上，并插入琼脂中的小垂直孔中，后端先插入。将含有包埋胚胎的平板以3000 g离心30 min。琼脂支持胚胎并使它们保持在确定的方向。然后，用钝针将胚胎从琼脂中挖出来。复盖作用将主要的细胞器分成不同的层，这种分层很容易通过明场显微镜观察到。许多荧光标记可用于确认活胚胎中成功分层。与某些细胞器相关的蛋白质将富集在特定的层中，表明共定位。可以回收单个卵子进行生化分析或移植到捐赠卵子中。该技术适用于其他大细胞中的细胞器分离，包括不同物种的卵和卵母细胞。
A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000 g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.