SEPARATION OF MURINE SPLENIC B-LYMPHOCYTE AND T-LYMPHOCYTE FOR USE INIMMUNOTOXICOLOGICAL STUDIES

The effects of xenobiotics on the immune system are often diverse and can involve secondary effects on cell types other than those of primar y immune origin. Thus, to better understand the effects of chemicals a nd drugs on immunocompetence, exposure of immune cell types to xenobio tics and assessment of functional activity have been conducted under i n vitro culture conditions. Interpretation of data obtained using this approach is reliant on the known composition of the cell populations used in these assay systems as well as on the ability to obtain purifi ed and functionally viable cells from mixed primary cell suspensions. The objective of this study was to develop an effective and reproducib le means of separating murine splenic B- and T-cells into purified pop ulations free of monocyte/macrophage and polymorphonuclear cell (PMN) contamination. Cell adherence and discontinuous Percoll gradients in c onjunction with immunomagnetic negative selection or antibody-mediated complement lysis were used to obtain Band T-lymphocytes with routine purities of greater-than-or-equal-to 95% Ig+ B-cells or L3T4+/Lyt-2+ T -cells and containing less-than-or-equal-to 1 % monocytes/macrophages and PMNs. Additionally, the separation of either B- or T-cells by buoy ant density on Percoll gradients resulted in the isolation of two dist inct subpopulations of cells. These subpopulations were characterized as either small lymphocytes with a high buoyant density (d = 1.085) or lymphocytes that were larger in size with a medium buoyant density (d = 1.075). Both subpopulations of either B- or T-cells were further fo und to consist of cells primarily in the G0/G1 stage of the cell cycle . All populations of isolated cells were found to be functionally resp onsive in vitro as evaluated using [H-3]thymidine incorporation follow ing stimulation with mitogens. This procedure provides an effective an d reproducible means of obtaining large numbers of purified and functi onally viable murine splenic B- and T-cells with minimal contamination by monocytes/macrophages and PMNs and should facilitate immunotoxicol ogical studies that are attempting to identify the immune cell type(s) targeted by a given xenobiotic.