Techniques for Purifying L-Cell Karyoplasts with Minimal Amounts of Cytoplasm
Cells may be enucleated by centrifuging monolayers of cells in the presence of the drug cytochalasin B (CB) (Prescott et al., 1972; Wright and Hayflick, 1972). These procedures allow one to obtain large numbers of enucleated cells (cytoplasts) and nuclei surrounded by a thin layer of cytoplasm containing an intact outer plasma membrane. The nuclear fragments were originally termed karyoplasts and described as being incapable of regeneration. Later it was reported that cytoplasts could be fused to karyoplasts using inactivated Sendai virus (Veomett et al., 1974). The resulting “reconstructed” cells are of potential use in many aspects of somatic cell studies in areas such as gene regulation, differentiation, and transformation (Shay, 1977; Shay and Clark 1979) (also see Chapter 19, this volume). A major difficulty in these experimental procedures has been the presence of a low percentage of cells in the cytoplast population which fail to enucleate and the more serious problem of a small fraction (approximately 6%) of the whole cells which detach during enucleation and thus contaminate the karyoplast population.
KeywordsFicoll Gradient Nuclear Donor Separation Chamber Differential Adhesion Exclude Trypan Blue
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