Separation and analysis of differentiating B lymphocytes from mouse spleens.

BALB/c spleen cells cultured for 72 hr in the presence of lipopolysaccharide (LPS) were separated into three different lymphoid cell populations on the basis of size by unit-gravity sedimentation through a linear albumin gradient. The small-cell region at the top of the gradient consisted of small lymphocytes positive for immunoglobulin light chains as shown by immunofluorescence. The intermediate-cell region in the middle portion of the gradient was composed of mitotic and intermitotic immunoblasts. Up to 90% of these cells possessed IgM, but only a very small amount of secreted IgM was detectable. The large-cell region at the bottom of the gradient contained mature plasma cells which secreted large amounts of IgM. No significant IgG synthesis was detected in cells throughout the gradient. Analysis of gradients by autoradiography sequentially during LPS stimulation showed that small lymphocytes are the primary cells responding to LPS. These cells synthesize DNA and then differentiate to become immunoblasts. The resulting immunoblasts then undergo several cycles of replication with a high percentage of these cells continuing the differentiation process to become mature plasma cells.