A preparative method for obtaining enucleated mammalian cells.

A PREPARATIVE METHOD FOR OBTAINING ENUCLEATED MAMMALIAN CELLS Michael H. Wigler and I. Bernard Weinstein Institute of Cancer Research and Departments of Medicine and Microbiology, College of Physicians and Surgeons Columbia University, New York, New York 10032 Received February 3, 1975 SUMMARY Mouse L cells can be enucleated in suspension by centrifugation in discontinuous Ficoll density gradients while in the presence of Cytochalasin B. Greater than 50% of the cytoplasts thus obtained attach to glass or plastic and undergo morphologic recovery within 2-4 hours of replating. Protein synthesis in cytoplasts undergoes a biphasic decay from an initial rate of approximately 50% of control nucleated cells. This method can yield up to 5 x 108 cytoplasts with consistently low levels of contamination by nucleated cells (less than 0.2%), and is well suited for obtaining quantitative amounts of cytoplasts or karyoplasts for physiologic or biochemical studies. INTRODUCTION In 1967 Carter reported that treatment of mouse L cells with the mold metabolite Cytochalasin B (CB) caused profound morphologic effects, including nuclear extrusion and, in a small number of cells, enucleation (i). Since that time, methods utilizing CB have been developed for obtaining large numbers of enucleated cells, or cytoplasts. According to those methods, when cells adhering to glass or plastic coverslips are inverted in media containing CB and centrifuged, the cells which remain attached are predominantly cytoplasts (2,3). The yields obtainable from such methods are limited by the total surface area which can be conveniently centrifuged, and are insufficient for many purposes. We have explored, therefore, the possibility that cells can be enucleated by centrifugation in isoosmotic density gradients where the theoretical limit of yield is determined by the band capacity of the gradient.