Improved methods for purification and depletion of monocytes from bovine peripheral blood mononuclear cells. Functional evaluation of monocytes in responses to lectins.
We have compared different techniques for the enrichment and depletion of monocytes from bovine peripheral blood mononuclear cells. Adherence to plasma-coated gelatin was the most efficient and reproducible method for enrichment of monocytes (80% monocytes), whereas depletion of peripheral blood mononuclear cells of monocytes (0.3% monocytes and less) was best achieved by defibrination of the blood from which the PBM were separated. In both instances, purity of the cell population could be improved further by an additional step, namely, FACS sorting with a monocyte-specific monoclonal antibody to purify monocytes (97% monocytes and more), and adherence to polystyrene to remove residual monocytes from defibrinated PBM (0.1% monocytes and less). Depletion of monocytes abolished the response of PBM to concanavalin A and phytohaemagglutinin. The lectin-induced response could be restored by adding gelatin/plasma purified monocytes. This activity of monocytes could be replaced by 2-mercaptoethanol.
Goddeeris
B M
BM
Baldwin
C L
CL
ole-MoiYoi
O
O
Morrison
W I
WI
D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D002448:Cell Adhesion; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D003208:Concanavalin A / Q000494:pharmacology*; D005260:Female; D007958:Leukocyte Count; D008213:Lymphocyte Activation; D008297:Male; D009000:Monocytes / Q000276:immunology; D010641:Phenotype; D010835:Phytohemagglutinins / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D010835:Phytohemagglutinins; D003208:Concanavalin A