Separation of rabbit marrow precursor cells by combined isopycnic sedimentation and electronic cell sorting

Abstract

Separation of developing cells into fractions of differing stages of maturity is critical to effective biochemical study of the process of cellular differentiation. Density gradient techniques utilizing rate-zonal or isopycnic separations have permitted partial separations based on cell mass or cell density. In this study the separation of various rabbit marrow cells was improved by high-speed flow analysis and sorting in a Coulter Two-Parameter Cell Sorter. After preliminary isopycnic separation of marrow cells in Ficoll-Hypaque, cells were sorted into lymphoid and myeloid elements, utilizing light-scatter (LS) profiles to determine sorting. Characteristic LS patterns were present for erythrocytes, lymphocytes, devitalized cells, and granulocytes. When erythrocytes and their precursors were removed by hypotonic lysis, the remaining granulocytes could be sorted to give samples with much greater purity with respect to developmental stage than was possible with density gradients alone. Thus the combination of techniques represents a significant improvement in the ability to study the mechanisms of normal or altered cellular maturation.