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bioRxiv · 10.1101/112334

High quality frozen extracts of Xenopus laevis eggs reveal size-dependent control of metaphase spindle micromechanics

Takagi, J.·Shimamoto, Y.
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Abstract

Cell-free extracts from unfertilized Xenopus laevis eggs offer the opportunity for a variety of biochemical assays for analyzing essential cell cycle events, such as metaphase spindle assembly. However, extracts utility is often hampered by their short storage-stability duration and high quality variation. Here, we report a simple two-step method for preparing frozen egg extracts that retain spindle assembly activity levels that are close to those of freshly prepared extracts. Extract degradation associated with the freeze-thaw process can be substantially reduced by using centrifugal filter-based dehydration and slow sample cooling. Large amounts of frozen extract stocks from single batch preparations allowed us to collect a large number of data in micromanipulation experiments that are intrinsically low-throughput and, hence, clarify correlations between metaphase spindle size and stiffness. We anticipate that our method provides an assay platform with minimized biological heterogeneity and makes egg extracts more accessible to researchers as distributable material.\n\nSummaryThe authors describe a method for preparing frozen extracts of Xenopus laevis eggs that retain spindle assembly activity levels that are close to those of freshly prepared extracts. This allowed for clarifying the correlation between spindle size and stiffness.

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Takagi, J., Shimamoto, Y.. 2017-02-28. High quality frozen extracts of Xenopus laevis eggs reveal size-dependent control of metaphase spindle micromechanics. https://doi.org/10.1101/112334

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