Isolation of Mitotic Spindles from Chinese Hamster Ovary Cells

Mitotic spindles were isolated from Chinese hamster ovary cells with an isolation medium that contained 5% glycerol, 1% dimethyl sulfoxide, 5% polyethylene glycol, 5 mM piperazine-N,N'-bis(2-ethanesulfonic acid), 1 mM ethylene glycol bis(γ-aminoethylether)tetraacetic acid, 0.5 mM MgS04, 0.5% Triton X-100, 0.5 mM GTP and 0.5 mM dithiothreitol at pH 6.8. The chromosomes generally were lost from the preparation; thus, electron microscopy of whole-mount preparations showed isolated spindles with a microtubule array in the spindle that attached to the centrosomes at each pole. An increase in the concentration of MgSO4 to 5 mM stabilized the chromosomal structure, and mitotic spindles with chromosomes attached in almost the same configuration as in vivo could be isolated. A general discussion of the procedures for isolating mitotic spindles from tissue cultured cells is included.