Enrichment of T lymphocytes from bovine peripheral blood mononuclear cells using an immuno-affinity depletion technique ("panning").

A simple and efficient method to enrich bovine T lymphocytes from peripheral blood mononuclear cells (PBMC) by immuno-affinity depletion ("panning") has been developed. The PBMC were initially separated by density gradient centrifugation on Histopaque of density 1.077 g/ml. The T lymphocyte subset was then separated from PBMC by depletion of membrane immunoglobulin (Ig) bearing cells which had an affinity for anti-Ig antibodies bound to polystyrene tissue culture flasks. An average of 95% of the nonadherent "panned" cells were identified as T lymphocytes using a label of peanut agglutinin conjugated with fluorescein isothiocyanate (PNA-FITC). Two percent of the PNA negative cells were Ig bearing cells. The average yield was 50% of the original T lymphocytes found in the PBMC population, and the cell viability as assessed by trypan blue exclusion was greater than 95%. The separation took approximately 2 hours, and the total number of T lymphocytes recovered from 40 ml of blood was in the range of 20-40 X 10(6).