Isolation of highly purified lymphocyte subsets for functional studies by means of an indirect rosette technique.

An indirect rosette assay, utilizing ox erythrocytes (RBC) coupled with rabbit anti-mouse IgG and lymphocytes sensitized with monoclonal mouse antibodies against membrane markers, was used for purification of lymphocyte subsets that were functionally intact. Either peripheral blood mononuclear cells (PBMC) or T lymphocytes isolated by sheep RBC rosetting could be used as starting material for obtaining pure T-cell subsets (T4 or T8). The following steps of the method were evaluated: the procedure of coupling rabbit anti-mouse IgG to ox RBC via the CrCl3 method, the experimental conditions for specific rosetting, and the use of Percoll for the separation of rosettes from the non-rosetting cells. Under optimal experimental conditions the recovery of positively selected cells was 45-55% of the cells originally present in the PBMC. The purity of these cells reached a value of more than 95%, whereas the contamination of the depleted fraction was less than 3%. The functional integrity, manifesting itself as proliferation after mitogen stimulation and as regulatory influences on in vitro Ig synthesis, appeared to be unimpaired. The described technique may be applied to the purification of various cell subpopulations for functional studies, provided monoclonal antibodies against membrane antigens are available.