Fractionation of differentiating cells using density perturbation

This paper describes the development of a new method for the fractionation of purified subpopulations of partially differentiated cells on continuous isopycnic gradients, using a density perturbation method based on the abili ty of cells to bind dense antibody-coated beads. Until now none of the avai lable fractionation techniques, such as magnetic cell fractionation has bee n efficient for separating subpopulations of partially differentiated cells . The fractionation experiments described in this report used promyelocytic HL-60 and DMSO-induced granulocytic HL-60 cells as a model system. populat ions of cells, modified by the binding of dense beads were fractionated on isotonic, isopycnic Optiprep gradients by centrifugation at 220Xg for 90 mi n at 20 degrees C. Examination of the different gradient fractions showed t hat, as cells bind increasing numbers of beads, they are found in the dense r regions of the isopycnic gradients. indirect immunofluorescence was combi ned with flow cytometric techniques to characterise the fractionation of pa rtially differentiated cells, flow cytometric results confirmed that as ant igenic determinants appear on the surface at higher levels of expression, t he number of bends binding to each cell increased. Furthermore, after fract ionation, when the bead-bound and non-bead-bound cells were cultured in the presence of DMSO, those cells that had bound more beads targeted to differ entiated cells were found to achieve terminal differentiation faster than t hose cells that had not been associated with any beads. (C) 2000 Elsevier S cience B.V. All rights reserved.