System for enumerating and separating bovine lymphocytes, using coordinated En and Ea markers: evaluation of lymph node lymphocytes.

A procedure was developed for enumeration and separation of bovine lymph node T-lymphocyte subpopulations. An evaluation was made of T-lymphocyte rosetting with sheep erythrocytes treated with aminoethylisothiouronium bromide and neuraminidase (Ea and En) in RPMI and L-15 media. The effect of holding the lymphocytes in culture over a period of 7 days on the En and Ea markers was established. The optimal period of incubation at 0 C for isolation of the rosetting populations with respect to initial rosette formation, enrichment of the rosetting population in the recovered cells, viability after incubation, and recovery of the cells after the separation was determined. An estimate of the overlap of the Ea and En populations also was made from the separation data. We found that approximately 32% of the lymph node lymphocytes marked with En and Ea rosettes, 11% with only En, and 27% with only Ea.