An improved technique for separating rosetted from non-rosetted lymphocytes

An improved technique for separating rosetted from non-rosetted lymphocytes is described. The major advantage of this new technique is the elimination of the need to mechanically resuspend the cell pellet resulting from the rosette formation step before using a density gradient to separate the rosetted from the non-rosetted cells. Instead the rosette containing cell pellet is directly overlayed with Percoll at a density of 1.078 g/ml and during a subsequent centrifugation the non-rosetted cells float to the surface of the Percoll while the rosetted cells remain in the cell pellet. The utility of this technique is illustrated by separating human T lymphocytes forming rosettes with neuraminidase treated sheep erythrocytes (En) from other mononuclear cells obtained by Ficoll-Hypaque separation of whole blood. Using several markers the resulting T cells were contaminated with less than 0.3% monocytes and 2% B cells. The non-T cell population contained less than 3% T cells. This method offers a rapid and easily reproducible means for obtaining highly purified cell populations.