Frozen egg extracts that retain spindle assembly activity (Takagi and Shimamoto, 2017)

Protocol from "High-quality frozen extracts of Xenopus laevis eggs reveal size-dependent control of metaphase spindle micromechanics" Takagi and Shimamoto, Mol. Biol. Cell August 1, 2017 vol. 28 no. 16 2170-2177.

Link to journal and full article: http://www.molbiolcell.org/content/28/16/2170.long

Preparation of Xenopus egg extracts

Cell-free extracts from unfertilized Xenopus laevis eggs arrested at metaphase were prepared as described previously (Desai et al., 1999), except that Energy Mix was used at 50× instead of 20×. For the preparation of filtered frozen extracts, freshly prepared extracts were loaded into centrifugal filter devices (200 µl per device; UFC510024; Millipore) and spun at 17,000 × g for 10 min at 4°C using a tabletop centrifuge (5424R; Eppendorf). The filtration was completed within 30 min after fresh extract preparation, as a prolonged incubation markedly reduced the quality of frozen extract stocks. Following centrifugation, the filter devices were placed upside down in collection tubes and spun at 2000 × g for ∼10 s (Multi Spin; TOMY) to transfer the concentrate to the tubes. The concentrate and flow-through fractions were then transferred to separate 1.5-ml test tubes by using low-adhesion pipette tips. The tubes were placed in a tube cooler (Chillette 12; Denville Scientific), which was precooled to 4°C, and frozen at a cooling rate of approximately −1°C/min in a −80°C freezer. Before the spindle assembly reaction, the frozen concentrate and flow-through fractions were thawed on ice and then combined by reintroducing the entire flow-through fraction to the concentrate fraction. The recovered extracts were used for assays following > 20 min of incubation on ice. Nonfiltered frozen extracts, which were used as controls in Figure 1, were prepared in the same manner as above, but without centrifugal filtration. For a part of the analysis shown in Figure 3, E–H, filter devices of a smaller mesh size (UFC501024; Millipore) were used. For the analysis shown in Supplemental Figure S2K, filter devices of a larger volume capacity (UFC210024; Millipore) and modified centrifugal parameters (5400 × g, 31 min) were used. The entire procedure was performed while keeping extract samples on ice as much as possible. Sample handling was performed using wide-bore pipette tips, which were prepared by cutting off the ends with a razor blade.

Preparation of spindle samples

Metaphase spindles were assembled by cycling the extracts with demembranated X. laevis sperm nuclei (∼400 nuclei/µl) once through interphase and back into metaphase (Desai et al., 1999). Each reaction was performed in a 20-µl volume at 16°C in 1.5-ml test tubes. Fixed samples were prepared 90 min after the start of spindle assembly, each with 4 µl of fixation buffer (125 mM HEPES, 2.5 mM EDTA, 2.5 M NaCl, 50 mM KCl, 25 mM MgCl2, 50 mM CaCl2, 10% formaldehyde, 60% glycerol, and 1 µg/ml 4′,6-diamidino-2-phenylindole [DAPI]) and 2 µl of extract reaction supplemented with 500 nM tetramethylrhodamine-tubulin, squashed between a clean slide and a 18 × 18 mm coverslip. Live spindle samples for micromanipulation experiments were prepared by performing the cycling reaction as described above, but with 500 nM tetramethylrhodamine-tubulin and 250 nM SYTOX Green.