M12.4, one of several B
lymphomas derived from BALB/c mice, was mutagenized with
ethyl methanesulfonate in vitro. M12.4.1, a subline of the mutant cells, was sensitive to
hypoxanthine-
aminopterin-
thymidine selective medium and was fused with normal splenic B lymphocytes of C57BL/6ByJ mice. The hybridomas obtained from this fusion were shown to express mu heavy chain, H-2KbDb, Iad, and Iab on the cell surface by analyses of flow microfluorometry and a cytotoxicity assay, although parental M12.4.1 lacked mu heavy chain on the cell membrane. These results demonstrate that the B cell hybridomas with B
cell surface antigens have been established in vitro.
IgM molecules on the cell surface of the hybridomas were shown to originate from normal B cells of C57BL/6ByJ mice by flow microfluorometry analyses after staining with
fluorescein-labeled Bet 1, a monoclonal rat antibody that recognizes Igh-6.5, a mouse
IgM allotypic determinant. These hybridomas could generate
IgM-secreting cells at the high frequency (more than 10% of the cultured cells) when stimulated with bacterial
lipopolysaccharide. On the other hand, parental M12.4.1 did not develop any
IgM-secreting cells under the same conditions. These findings suggest that these B cell hybridomas with B
cell surface antigens may be a good model for the study of B cell differentiation.