Enrichment of human bone marrow mononuclear phagocytes and characterization of macrophage subpopulations by immunoenzymatic double staining.
In order to isolate and enrich bone marrow mononuclear phagocytes, we performed magnetic-activated cell sorting using beads coupled to a monoclonal antibody directed against the monocyte/macrophage surface molecule CD14. Colocalization of antigens in single cells was achieved by combining an alkaline phosphatase-anti-alkaline phosphatase and an avidin-biotin complex immunoassay, avoiding the use of peroxidase. Bone marrow macrophages were first labelled by the monoclonal antibody PG-M1 (anti-CD68). Subsequently, cytoplasmic and/or surface double staining by the monoclonal antibodies against HLA-DR and Mac-2 antigen or the lectin GSA-I-B4 was carried out. Whereas HLA-DR was co-expressed by the great majority of PG-M1+ macrophages (84.9%+/-6.9%), only a subpopulation exhibited Mac-2 (69.9%+/-5.9%) antigen or galactoside structures detected by GSA-I-B4 (65.0%+/-6.7%). The latter result differed only slightly from the percentage of GSA-I-B4+ macrophages determined in a previous comparative immunomorphometrical study. Therefore, using our method of isolation and enrichment by magnetic-activated cell sorting, only a negligible portion of macrophages is apparently stimulated, as shown by GSA-I-B4 staining. This methodology seems to be a valuable tool for further studies on the monocyte-macrophage system.
Baldus
S E
SE
Institute of Pathology, University of Cologne, Germany.
Wickenhauser
C
C
Stefanovic
A
A
Schmitz
B
B
Thiele
J
J
Fischer
R
R
D015703:Antigens, CD / Q000096:biosynthesis; D015214:Antigens, Differentiation, Myelomonocytic / Q000096:biosynthesis; D015415:Biomarkers; D001854:Bone Marrow Cells / Q000166:cytology* / Q000276:immunology; D002469:Cell Separation / Q000379:methods*; D006801:Humans; D007124:Immunoenzyme Techniques; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000276:immunology; D008264:Macrophages / Q000166:cytology* / Q000276:immunology; D010586:Phagocytes / Q000276:immunology; D013194:Staining and Labeling / Q000379:methods*; D000097382:CD68 Molecule
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D015415:Biomarkers; C067980:CD68 antigen, human; D000097382:CD68 Molecule