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Journal of Histochemistry and Cytochemistry, Vol. 49, 219-228, February 2001, Copyright © 2001, The Histochemical Society, Inc.

ARTICLE

Identification and Isolation of Rat Bone Marrow-derived Mast Cells Using the Mast Cell-specific Monoclonal Antibody AA4

Maria Célia Jamura, Ana Cristina G. Grodzkia, Andrea N. Morenob, Luciana de Fátima C. de Mellob, Maria Veronica D. Pastora, Elsa H. Berensteinc, Reuben P. Siraganianc, and Constance Olivera
a Departamento de Morfologia, Faculdade de Medicina de Ribeirão Preto–Universidade de São Paulo, Ribeirão Preto, Brasil
b Departamento de Biologia Celular, Universidade Federal do Paraná, Curitiba, PR, Brasil
c Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland

Correspondence to: Constance Oliver, Faculdade de Medicina de Ribeirão Preto, Depto. Morfologia, Av. Bandeirantes 3900, 14049-900 Ribeirão Preto, SP, Brasil. E-mail: coliver@rmf.fmrp.usp.br

Previous studies of mast cell maturation, structure, and function have been hampered by the lack of mast cell-specific markers. In this study, using a well-characterized mast cell-specific monoclonal antibody, MAb AA4, mast cells from rat bone marrow in various stages of maturation were isolated and characterized. The very immature mast cells, which have not been previously described, contained few granules and would not be recognized as mast cells by standard cytological methods. Pure populations of mast cells were isolated from the bone marrow using MAb AA4-conjugated magnetic beads. The same stages of maturation were observed in the isolated mast cells as were seen in the unfractionated bone marrow. All of these cells were immunopositive for the {alpha}-subunit of Fc{varepsilon}RI, IgE, and c-kit, confirming their identity as mast cells. By direct counting of immunolabled cells and by flow cytometry, approximately 2.4% of the cells in the bone marrow are mast cells. Staining with toluidine blue and berberine sulfate, as well as RT-PCR of the cells, indicates that these cells are connective tissue-type mast cells. The use of immunological methods for identification of mast cell precursors should facilitate the study of these cells. (J Histochem Cytochem 49:219–228, 2001)

Key Words: mast cells, gangliosides, maturation, bone marrow, immunochemistry, rat


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