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).
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D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology; D000915:Antibody Affinity; D002417:Cattle; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002499:Centrifugation, Density Gradient; D005260:Female; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D007074:Immunoglobulin G / Q000276:immunology; D007163:Immunosorbent Techniques; D037102:Lectins; D019887:Peanut Agglutinin; D013601:T-Lymphocytes; D013861:Thiocyanates
D000888:Antibodies, Anti-Idiotypic; D005452:Fluoresceins; D007074:Immunoglobulin G; D037102:Lectins; D019887:Peanut Agglutinin; D013861:Thiocyanates; D016650:Fluorescein-5-isothiocyanate