Quantitative flow cytometric and clonogenic evaluation of glass bead affinity fractionation of antibody-labeled murine bone marrow.
Monoclonal antibodies and a glass bead affinity fractionation technique were used to selectively deplete subpopulations of murine bone marrow cells. Flow cytometric analyses permitted quantitative measurement of subpopulation depletion and characterization (light scatter and fluorescence intensity) of both the eluted and bound cell subpopulations. Mouse bone marrow cells were labeled with selected monoclonal rat anti-mouse antibodies directed against cell surface antigens and were eluted through a glass bead column coated with goat anti-rat immunoglobulin. Unlabeled cells passed through the column, whereas cells labeled with the antibody were selectively retained. Column operating conditions for optimal depletion of labeled cells were determined. With specific column conditions, 97% of the antibody positive cells were retained on the column. In addition, clonogenic assays on cells sorted from unfractionated and column fractionated preparations provided estimates of the fraction of granulocyte macrophage progenitors (CFU-GM) in the different cell subpopulations. The enrichment of CFU-GM achieved in the eluted cell populations was dependent upon the antibody used for cell labeling and ranged from four- to six-fold. Since large numbers of cells can be processed rapidly, this technique, in combination with antibodies specific for non-clonogenic cells, is particularly suitable when preparations enriched in colony-forming progenitors are required.
Matsson
P
P
Biomedical Sciences Division, Lawrence Livermore National Laboratory, University of California 94550.
Pallavicini
M G
MG
Summers
L
L
D000818:Animals; D000906:Antibodies; D000954:Antigens, Surface / Q000276:immunology; D001853:Bone Marrow / Q000276:immunology; D001854:Bone Marrow Cells; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D003114:Colony-Forming Units Assay; D005434:Flow Cytometry; D005898:Glass; D007158:Immunologic Techniques; D051379:Mice; D008809:Mice, Inbred C3H
D000906:Antibodies; D000954:Antigens, Surface