Isolation of neutrophils and eosinophils from the peripheral blood of cattle and comparison of their functional activities

Abstract

A procedure for isolating purified populations of neutrophils and eosinophils from bovine peripheral blood is described. The procedure involves the centrifugation of anticoagulated whole bovine blood, flash lysis of the packed red blood cells and separation of the eosinophils from the neutrophils by centrifugation on a Ficoll-Hypaque density gradient. The following parameters were evaluated on populations of greater than 90% pure eosinophils and neutrophils: (1) random migration under agarose, (2) ingestion of 125I-labeled Staphylococcus aureus, (3) nitroblue tetrazolium reduction, (4) iodination, or the conversion of 125I to a trichloroacetic acid precipitable form, (5) antibody-dependent cell-mediated cytotoxicity, and (6) the amount of intracellular peroxidase. Neutrophils were significantly more active than eosinophils in the ingestion of S. aureus and in antibody-dependent cell-mediated cytotoxicity. Eosinophils were much more active than neutrophils in the resting and stimulated iodination test and they contained higher levels of peroxidase. There was no difference between the two cell types in their ability to reduce nitroblue tetrazolium or migrate under agarose. The effect of Ficoll-Hypaque on granulocyte function was determined. Previous contact with Ficoll-Hypaque significantly reduced random migration but other function tests were unaffected.