A two-step procedure for obtaining normal peripheral blood T-lymphocytes using continuous equilibrium density gradient centrifugation on percoll.

Equilibrium centrifugation of either peripheral blood mononuclear cells or of pure lymphocytes (obtained by carbonyl iron or glass bead adherence removal of monocytes) on a continuous density gradient of Percoll yielded lymphocyte fractions containing between 92 and 99% T lymphocytes as shown by sheep red blood cell rosetting. B lymphocytes with surface immunoglobulin were found in the regions of low density (1.03-1.065 g/ml) and T lymphocytes in the regions of higher density (1.06-1.08 g/ml). TM lymphocytes with their characteristic positive 'dot' pattern of staining for non-specific esterase were also found mainly in regions of high density. It was concluded that Percoll continuous equilibrium density gradient centrifugation can be used to obtain T lymphocytes in high yield, with high viability and without metabolic changes which may occur after contact with sheep red blood cells. The esterase staining suggested that there was also some separation of T lymphocyte subsets.