The concentration and resolution of primitive hemopoietic cells from normal mouse bone marrow by negative selection using monoclonal antibodies and Dynabead monodisperse magnetic microspheres.
High proliferative potential colony-forming cells (HPP-CFC) detected in clonal agar culture in the presence of the combined stimulus of colony-stimulating factor 1 (CSF-1) + interleukin 3 (IL-3) + interleukin 1 alpha (IL-1 alpha) are closely related to developmentally early progenitor cells capable of reconstituting the hemopoietic system of lethally irradiated mice following transplantation. Flow cytometric analysis and sorting of normal, unperturbed bone marrow has shown that HPP-CFC are B220- and 7/4-, whereas the committed progenitors of the macrophage lineage responsive to CSF-1 alone (CSFCSF-1) are B220- and 7/4+. Negative immunomagnetic selection using an anti-7/4, anti-B220 antibody cocktail and second-antibody-coupled Dynabead microspheres to replace flow cytometry results in the highly reproducible and specific enrichment of HPP-CFC, and simultaneous resolution of HPP-CFC from CFCCSF-1. The tenfold enrichment of HPP-CFC compared with unfractionated bone marrow cell suspensions was comparable to that obtained by fluorescence-activated cell sorting. Enrichment was achieved with negligible loss of HPP-CFC at the immunomagnetic bead selection step, and 65% of HPP-CFC were recovered. The method is rapid, highly reproducible, and efficient, and has wide application to the separation of rare hemopoietic cells from normal bone marrow.
Bertoncello
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I
Cell Biology Group, Peter MacCallum Cancer Institute, Melbourne, Victoria, Australia.
Bradley
T R
TR
Hodgson
G S
GS
D000818:Animals; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D001853:Bone Marrow / Q000276:immunology; D001854:Bone Marrow Cells; D002452:Cell Count; D002455:Cell Division; D002469:Cell Separation; D003114:Colony-Forming Units Assay; D005260:Female; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D008280:Magnetics; D051379:Mice; D008863:Microspheres; D011137:Polystyrenes; D051381:Rats
D000911:Antibodies, Monoclonal; D011137:Polystyrenes