Identification and isolation of rat bone marrow-derived mast cells using the mast cell-specific monoclonal antibody AA4

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 c ells from rat bone marrow in various stages of maturation were isolated and characterized. The very immature mast cells, which have not been previousl y described, contained few granules and would not be recognized as mast cel ls by standard cytological methods. Pure populations of mast cells were iso lated from the bone marrow using MAb AA4-conjugated magnetic beads. The sam e stages of maturation were observed in the isolated mast cells as were see n in the unfractionated bone marrow. All of these cells were immunopositive for the alpha -subunit of Fc epsilon RI, IgE, and c-kit, confirming their identity as mast cells. By direct counting of immunolabled cells and by flo w cytometry, approximately 2.4% of the cells in the bone marrow are mast ce lls. Staining with toluidine blue and berberine sulfate, as well as RT-PCR of the cells, indicates that these cells are connective tissue-type mast ce lls. The use of immunological methods for identification of mast cell precu rsors should facilitate the study of these cells.