Chapter 7 Enucleation of Mammalian Cells in Suspension

Prescott et al. reported an efficient, relatively simple technique for the enucleation of large numbers of cultured mammalian cells. The method entails plating cells on a glass coverslip or some other surface and centrifuging the surface, cell-side down in medium containing the mold metabolite cytochalasin B. Prescott's method or variations of it have found widespread application. The use of enucleated cells, or cytoplasts, thus prepared is justified by the findings that cytoplasts retain many of the morphological and behavioral characteristics of whole cells. The enucleation method of Prescott nevertheless has certain drawbacks. These include the fact that the yield is limited by the surface area that can be exposed to centrifugal forces, and the method cannot be used on cells that adhere poorly or not at all to solid surfaces. Authors have therefore explored the possibility that cells can be enucleated while in suspension by isopycnic centrifugation in the presence of cytochalasin B, and have developed a procedure of high efficiency and yield that can theoretically be applied to almost any eukaryotic cell lacking a cell wall.