Expression of antibody-forming cell clones in two-stage cultures: an attempt to separate proliferation and maturation events.

Peripheral blood lymphocytes (PBL) from non-immunized rabbits or from rabbits immunized several months previously with a single dose of sheep red blood cells (SRBC) were cultured at several different cell densities in the presence of antigen for 5 days. The cells were then distributed in 10 microliter microcultures at different cell densities to estimate the frequency of responding units. It was found that a shift up in cell concentration from first to second stage allows a more efficient expression of antibody-forming cell clones. We conclude that, during the first stage at low cell density, the precursor cells proliferate and, when they are partitioned in microcultures and the cell concentration is raised, maturation to antibody-forming cells occurs. This approach allows a separation of the proliferation event from the maturation event and it may prove to be a useful tool in the study of B-cell differentiation.