Cell-free cytoplasmic extracts prepared from Xenopus eggs have been used extensively to recapitulate and characterize intracellular events in vitro. Egg extracts can be induced to transit the cell cycle and reconstitute assembly of dynamic structures including the interphase nucleus and the mitotic spindle. In this protocol, methods are described for preparing crude cytoplasmic extracts from Xenopus eggs and embryos that are arrested in metaphase of the cell cycle. The basic protocol uses

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... ilized Xenopus laevis eggs, which are crushed by centrifugation in the presence of EGTA to preserve the natural cytostatic factor (CSF) activity that maintains high levels of Cdk1/cyclin B kinase and metaphase arrest. In the second method, the basic procedure is adapted for Xenopus tropicalis eggs with minor modifications to accommodate differences in frog size, timing of egg laying, and temperature and salt sensitivity. The third variation takes advantage of the synchronous divisions of fertilized X. laevis eggs to generate extracts from embryos, which are arrested in metaphase by the addition of nondegradable cyclin B and an inhibitor of the anaphase-promoting complex (APC) that together stabilize Cdk1/cyclin B kinase activity. Because they are obtained in much smaller amounts and their cell cycles are less perfectly synchronized, extracts prepared from embryos are less robust than egg extracts. X. laevis egg extracts have been used to study a wide range of cellular processes. In contrast, X. tropicalis egg extracts and X. laevis embryo extracts have been used primarily to characterize molecular mechanisms regulating spindle and nuclear size. MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at Dilute LPC protease inhibitor cocktail 1:1000 into CSF-XB. Prepare fresh and use immediately. This buffer is used for X. laevis eggs and embryos. CSF-XB solution #2 for X. tropicalis eggs This buffer is used for X. tropicalis eggs and contains additional EGTA and magnesium chloride. CSF-XB+ solution #2 (CSF-XB solution #2 supplemented with 10 µg/mL LPC) Dilute LPC protease inhibitor cocktail 1:1000 into CSF-XB solution #2. Prepare fresh and use immediately. This buffer is used for X. tropicalis eggs and contains additional EGTA and magnesium chloride. Cytochalasin D or B (10 mg/mL, Sigma-Aldrich C8273 or Sigma-Aldrich C6762, respectively) Dissolve in DMSO and store in 50 µL aliquots at −20˚C. Dejelly solution #1 for Xenopus laevis eggs Dejelly solution #2 for Xenopus tropicalis eggs and Xenopus laevis embryos Unlike the X. laevis dejelly solution, the X. tropicalis buffer does not contain salts. Dimethyl sulfoxide (DMSO) (anhydrous, high purity such as Sigma-Aldrich) Energy mix (50×) Note that some labs do not add energy mix to egg extracts.