A rapid rosette technique for quantitation and separation of mononuclear cell subsets using monoclonal antibodies

We present a rapid and simple method for simultaneous quantitation and separation of mononuclear cell (MNC) subsets. When lymphoid cells are sensitized with monoclonal antibodies of the OK and Leu series, they rapidly form rosettes with ox erythrocytes (ORBC) coated with affinity-purified rabbit IgG against mouse IgG. Rosette-forming cells (RFC) may then be counted and separated from non-rosetting MNC by Isopaque-Ficoll gradient centrifugation. The yield and viability are close to 100% after ORBC lysis. Adherent cells do not interfere. Isolated T8+ and Leu3a+ cells were further tested: the purity was 97-99%, and the cells were functionally intact with respect to their modulating activity on the generation of immunoglobulin-secreting cells by MNC after stimulation with pokeweed mitogen.