Separation of lymphocyte subpopulations using biotin-avidin erythrocyte rosettes

The major method for isolating murine lymphocyte subpopulations involves negative selection using antibody plus complement-mediated cytolysis. We have developed an efficient rosette method for enriching murine B and T cells using biotin-conjugated antibodies and avidin-coated sheep erythrocytes. Rosetted and non-rosetted subpopulations are separated rapidly on Percoll cushions. In systems employing rabbit anti-mouse immunoglobulin or monoclonal rat anti-mouse Thy-1.2 conjugated to biotin, positively-selected cells are greater than 90% pure while negatively-depleted populations possess less than 2% contamination with unwanted cells. Recoveries from starting spleen cell populations range between 50 and 75%. This method provides an easily performed alternative for obtaining positively and negatively selected cell populations and can be used with any biotin-conjugated antibody protein.