Enrichment of murine marrow cells for progenitors of multilineage hemopoietic colonies.
We attempted to develop a purification method for cell cycle-dormant murine multipotential progenitors that is simple and has a high recovery. Multilineage colony formation from mice that had been treated with 150 mg/kg 5-fluorouracil was assayed in cultures containing interleukin-3, interleukin-6, and erythropoietin. The cells with density 1.0631-1.0770 g/cm3 were approximately 40-fold enriched for multipotential progenitors. Negative immunomagnetic selection with a panel of lineage-specific monoclonal antibodies including anti-B220, anti-Gr-1, anti-Mac-1, anti-L3T4, and anti-Lyt-2 resulted in a cumulative 150-fold enrichment. When the density-separated and lineage-negative cells were further enriched by fluorescence-activated cell sorting with monoclonal antibody D7 (anti-Ly-6A/E), total enrichment averaged 800-fold and recovery was 17%. The method described here provides a relatively simple technique for the isolation of the multipotential progenitors and should be useful for the studies of regulation of hemopoiesis.
Shih
J P
JP
Department of Medicine, Medical University of South Carolina, Charleston.
Zeng
H Q
HQ
Ogawa
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D001854:Bone Marrow Cells; D002452:Cell Count; D002453:Cell Cycle; D002469:Cell Separation; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D003470:Culture Media; D004921:Erythropoietin / Q000494:pharmacology; D005260:Female; D005434:Flow Cytometry; D005472:Fluorouracil / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D007377:Interleukin-3 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains
D000911:Antibodies, Monoclonal; D003470:Culture Media; D007377:Interleukin-3; D015850:Interleukin-6; D004921:Erythropoietin; D005472:Fluorouracil