Fractionation of differentiating cells using density perturbation

• Published in: Journal of immunological methods 2000-06, Vol.240 (1), p.93-99
• Main Authors: Bildirici, L, Rickwood, D
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Cell Differentiation; Cell fractionation; Cell Separation - methods; Centrifugation, Isopycnic - methods; Density perturbation; Differentiating cells; Diverse techniques; Fundamental and applied biological sciences. Psychology; Granulocytes - cytology; HL-60 Cells - cytology; Humans; Immunomagnetic Separation - methods; Molecular and cellular biology; Surface antigens
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(00)00180-0

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 ability of cells to bind dense antibody-coated beads. Until now none of the available fractionation techniques, such as magnetic cell fractionation has been 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. Populations of cells, modified by the binding of dense beads were fractionated on isotonic, isopycnic Optiprep gradients by centrifugation at 220× g for 90 min at 20°C. Examination of the different gradient fractions showed that, as cells bind increasing numbers of beads, they are found in the denser regions of the isopycnic gradients. Indirect immunofluorescence was combined with flow cytometric techniques to characterise the fractionation of partially differentiated cells. Flow cytometric results confirmed that as antigenic determinants appear on the surface at higher levels of expression, the number of beads binding to each cell increased. Furthermore, after fractionation, 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 differentiated cells were found to achieve terminal differentiation faster than those cells that had not been associated with any beads.