Enrichment of human bone marrow mononuclear phagocytes and characterization of macrophage subpopulations by immunoenzymatic double staining

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Abstract

Summary

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. Co-localization 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-B+4 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.

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