Centrifugal Elutriation

Abstract

Centrifugal elutriation is a velocity sedimentation method that separates cells on the basis of size, shape, and density. Lindahl first used this technique to separate cell subpopulations from a variety of biological systems (Lindahl 1956). Since cell volumes increase from G1 through G2/M phases of the cell cycle (Anderson and Petersen 1964; Steen and Lindmo 1978), synchronous mammalian cell populations in each phase of the cell cycle can be isolated by this separation method (Meistrich et al. 1977a, b; Keng et al. 1980). Several advantages are provided by centrifugal elutriation in obtaining synchronous cells. Using approximately 10⁹ cells, between 10⁷ and 10⁸ synchronized G1, S and G2/M cells can be obtained by this method in a short period of time (less than 1 h). There is a high degree of homogeneity in the cell populations obtained by this method, usually 90–95% for G1 phase cells, 75–85% for S phase cells, and 60–75% for G2/M phase cells (Liu et al. 1989). Finally, centrifugal elutriation does not require an arrest of the cell cycle to obtain synchronized cells, and the entire separation procedure is performed in tissue culture medium. Since there is little or no perturbation to cell growth, this method can be used to establish the temporal sequence of cell cycle events. The utility of this method can be exemplified in the study of the roles of cyclin A (Marraccino et al. 1992; Pagano et al. 1992) and cyclin E (Koff et al. 1992) in the human cell cycle.