Large-Scale Enucleation of Mammalian Cells

This chapter describes a method for obtaining large numbers of enucleated cells and karyoplasts. This method utilizes 25 cm² tissue-culture flasks both as substrate for cellular growth and as centrifuge tubes. The method is described for mouse L cells, but it should be applicable to any cell that grows in monolayers. Cells are grown in the tissue culture flasks for at least 24 hours to allow the cells to attach firmly. The tissue culture medium is then removed, and medium containing cytochalasin B (CB) is added to the flask until it is completely filled. The cap of the flask is then replaced and tightened. The enucleation procedure is performed in a Sorvall RC-2B centrifuge, utilizing the GSA rotor, both of which are prewarmed to 37°C. Approximately 125 ml of water at 37°C is added to each rotor well; a filled flask is then added to each well. The percentage of enucleation after one centrifugation is good, generally greater than 95%. The karyoplasts form a pellet in the corner of the flask and are easily recovered.