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An improved negative immunomagnetic selection strategy has been devised for the enrichment of primitive hemopoietic cells using the high proliferative potential colony-forming cell (HPP-CFC) assay as an index of stem cell purification. Immunomagnetic selection was carried out using goat anti-rat conjugated M-450 Dynabeads and a cocktail of rat monoclonal antibodies directed against lineage antigens expressed on B-lymphocytes (B220), neutrophils and activated macrophages (7/4), differentiating erythroid cells (YW 25.12.7), and T-lymphocyte subsets (Lyt-2 and L3T4). This negative selection strategy results in the highly reproducible enrichment of HPP-CFC with negligible loss of HPP-CFC at the immunomagnetic selection step. A 30-fold enrichment of HPP-CFC stimulated by interleukin 3 (IL-3) plus colony-stimulating factor (CSF-1), or interleukin 1 alpha (IL-1 alpha) plus IL-3 plus CSF-1, is obtained with simultaneous resolution of HPP-CFC from progenitor cells of low proliferative potential responsive to CSF-1 alone (LPP-CFC). Flow cytometric analysis of these lineage-negative cells reveals that they almost exclusively exhibit the light-scattering characteristics of blast cells and the morphology of a candidate hemopoietic stem cell. Positive fluorescence-activated cell sorting of immunomagnetically pre-enriched normal bone marrow cells using wheat germ agglutinin yields cell preparations with a cloning efficiency of up to 45% and a HPP-CFC content of 20%.

Type

Journal article

Journal

Experimental hematology

Publication Date

02/1991

Volume

19

Pages

95 - 100

Addresses

Cell Biology Group, Peter MacCallum Cancer Institute, Melbourne, Victoria, Australia.

Keywords

Bone Marrow Cells, Hematopoietic Stem Cells, Bone Marrow, Animals, Mice, Inbred Strains, Mice, Colony-Stimulating Factors, Interleukin-3, Interleukin-1, Antibodies, Monoclonal, Flow Cytometry, Cell Separation, Immunologic Techniques, Magnetics