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

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Baldus, SE; Wickenhauser, C; Stefanovic, A; Schmitz, B; Thiele, J; Fischer, R

Published Date

  • April 1998

Published In

Volume / Issue

  • 30 / 4

Start / End Page

  • 285 - 291

PubMed ID

  • 9610820

International Standard Serial Number (ISSN)

  • 0018-2214

Digital Object Identifier (DOI)

  • 10.1023/a:1003268008228

Language

  • eng