Long-term culture of normal mouse B lymphocytes

A procedure is described for the preparation of long-term lines of normal mouse B lymphocytes. Surface immunoglobulin-bearing splenic B lymphocytes were purified with the fluorescence-activated cell sorter and then cultured with lipopolysaccharide for 1-4 wk. The cells were then transferred into medium supplemented with a T-hybridoma-derived supernatant containing interleukin 2 (IL2). Continuous feeding with this supernatant led to the establishment of cell lines that also could be propagated to IL 2-free medium containing interleukin 1 but not in culture medium alone. Cell lines have been propagated in this manner for as long as 10 mo. The cells in these lines have the appearance for small, dense lymphocytes, which all bear surface IgM detectable by immunofluorescence, rosetting, and surface radiolabeling and immunoprecipitation. The cells express Ia and lack Thy 1. These cultured B lymphocytes are unresponsive to lipopolysaccharide but can be activated to become more rapidly dividing, immunoglobulin-secreting cells by exposure to culture supernatants containing both T-cell-replacing factor and IL 2.