ra ins of Mice at Different Ages

We have recently been able to improve the conditions for in vitro lymphocyte cultures such that every growth-inducible murine B cell, in the presence of a Bcell mitogen, will initiate growth and maturation to IgM-, IgG-, and IgAsecreting plaque-forming cells (PFC) (1, 2). 1 This was achieved mainly by adding 2-mercaptoethanol (3), a growth-supporting fetal calf serum (4), growthsupporting thymus cells (2), and a B-cell mitogen (4-6) to the cultures. The presence of thymus cells, in particular, yielded a large increase in the number of growth-initiating B cells. We found that every dividing B cell within a clone, stimulated by mitogen, secreted Ig (2). A modified hemolytic plaque assay with protein A-coated sheep erythrocytes (SRC) and anti-Ig antisera as developing antibodies was employed to detect every Ig-secreting cell as a plaque (7). These culture conditions and the plaque assay for the detection and enumeration of all stimulated, secreting B-cell clones made it possible to do limiting dilution analyses of the number of bacterial lipopolysaccharide (LPS)-reactive B cells. Every third B cell in these spleen cell preparations of 6to 8-wk old C3Hfrif mice proved to be LPS-reactive yielding a clone of IgM-secreting PFC (2). The capacity of a given lymphocyte population to respond to a B-cell mitogen can, therefore, be assessed by limiting dilution analyses in vitro and can be expressed as frequencies of reactive B cells. In this paper we determine the frequencies of mitogen-reactive B cells yielding an IgM-PFC response mainly to the mitogen LPS (5) and in some cases also to the mitogen lipoprotein (6) in spleen, lymph node, bone marrow, thymus, thoracic duct, and peripheral blood of several inbred strains of mice at different ages.