Measurement of phagocytosis and oxidative burst of canine neutrophils: high variation in healthy dogs.

Quantitative analysis of phagocytosis and oxidative burst in canine polymorphonuclear (PMN) cells was performed by flow cytometry techniques. Different concentrations of phorbol myristate acetate (PMA) were used to modulate PMN phagocytosis. A low concentration of PMA (3 nmol) resulted in increased phagocytic activity of canine PMN, which could not be enhanced by higher dosages. Experiments with a reference cell population showed high losses of PMN, most probably by adherence to plastic material. It was possible to avoid this loss by layering all ingredients on cushions of Histopaque. However, Histopaque had a negative influence on the phagocytic activity of canine PMN. The use of PMA led to a dosage-dependent increase in the oxidative burst measured by the production of reactive oxygen species (ROS). Cushions of Histopaque were used to avoid cell loss. There was no negative influence of Histopaque on ROS formation. Storage of canine PMN for 24 h at room temperature had no negative influence on phagocytosis or oxidative burst measurements. Variations in the ROS assays conducted by two different examiners could be eliminated by use of a Histopaque-cushion.