The separation of different cell classes from lymphoid organs. X. Preparative electrophoretic separation of lymphocyte sub‐populations from mouse spleen and thoracic duct lymph

Conditions have been established for the separation of viable mouse lymphoid cells by continuous free‐buffer film preparative electrophoresis. The detailed electrophoretic distribution profiles of T and B lymphocytes from mouse spleen and thoracic duct have been determined. Cell surface θ‐antigen was used as a marker for T cells, and high surface‐density of immunoglobulin as a marker for B cells. Spleen cells from athymic “nude” mice were also studied. In the unselected normal spleen cell populations B lymphocytes are heterogeneous, about 60% being of low mobility with the remainder distributing broadly, and extending into the highest mobility fractions. T lymphocytes are predominantly of high mobility. Lymphoid cells lacking markers of either the B or T lineage are of intermediate mobility. There is only partial separation of T and B cells because of the extensive overlap between the populations. The high mobility B cells, which separate along with T cells, include a substantial proportion of large cells, and include cells with high surface density of immunoglobulin. The majority of these large B cells can be selectively eliminated by their adherence on passage through a glass‐bead column. By pre‐selecting the 50% non‐adherent lymphocytes from spleen as the starting material, a very sharp and more extensive separation of B and T cells can be achieved, with 100% pure B cells and 90% pure T cells in many fractions. However these samples are not representative of the total T and B cell populations of spleen. In thoracic duct lymph high mobility B‐cells are absent, there is little overlap between T and B cell mobility. 100% pure T and B cells can be isolated.