Analysis of murine lymphocyte subpopulations by dual-color flow cytometry: Technical considerations and specificities of monoclonal antibodies directed against surface markers

We performed detailed phenotypic analysis of murine lymphocytes from thymus, spleen, lymph node, and peripheral blood using commercially available monoclonal antibodies, each with specifities for membrane surface markers and dual-color flow cytometry. Erythrocyte lysis techniques were utilized for lymphocyte preparation so that inherent difficulties with gradient techniques would be avoided, such as the potential for loss of abnormally sized cells. These studies demonstrated that the specifities of each monoclonal must be carefully determined; for example, the Lyt-1 monoclonal, frequently utilized to identify helper/inducer T cells, also reacts with suppressor/cytotoxic (Lyt-2⁺) cells; helper/inducer cells are better studied with a more recently available monoclonal, L3T4. Cells from different tissues may differ greatly not only in the presence of surface markers, but also in the surface density of each marker; this density can be studied and quantitated using appropriate analytic software. We also show that larger and more granular lymphocytes appear to be enriched for surface Ia antigen, indicating that these cells may be activated or regulatory subsets; these large, Ia⁺ T-cells will be lost from analysis if standard, narrow gate settings are used for analyzing forward and side-scatter characteristics or for cell sorting.