Techniques for purifying L-cell karyoplasts with minimal amounts of cytoplasm.
Mouse L-929 cells centrifuged in the presence of cytochalasin B (CB) separate into cytoplasmic (cytoplasts) and nuclear (karyoplasts) fractions, but both of these fractions contain some whole-cell contaminants. We describe here two techniques for eliminating most of these whole cells from the karyoplast population. The first involves incubating cells in the presence of tantalum (Ta) particles, followed by enucleation and separation, on a Ficoll gradient. The small karyoplasts separate from the whole cells which contain a greater number of Ta particles. The second procedure uses a cell sorter that allows purification of karyoplasts from whole cells on the basis of light scatter. Both methods remove the whole-cell contaminants and the larger, and possibly viable, karyoplasts from the smaller karyoplast population. These purified karyoplasts are unable to regenerate but may be rescued by fusion with cytoplasts.
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002467:Cell Nucleus; D003102:Colloids; D003571:Cytochalasin B; D005362:Ficoll; D007739:L Cells / Q000648:ultrastructure*; D051379:Mice; D013635:Tantalum
D003102:Colloids; D005362:Ficoll; D003571:Cytochalasin B; D013635:Tantalum