An improved method to quantitate phagocytosis with mineral oil particles which avoid cell flotation

Abstract

We have shown that actively phagocytosing human polymorphonuclear leukocytes (PMN) float on top of the incubation medium when Oil Red O containing paraffin oil particles (density 0.8740) are used as the phagocytosable material. This implies that the quantitation of phagocytosis based on the recovery of Oil Red O in phagocytosing cells pelleted after centrifugation would be underestimated. We therefore prepared particles of progressively increasing density by mixing paraffin oil (density 0.8740) with silicon oil (density 1.0802). Cell flotation also occured with paraffin oil-silicon oil particles and could be avoided only when the density of the particles used had reached 0.9952 g/cm 3. Paraffin oil-silicon oil particles of a density sufficient to dissolve the dye Oil Red O were therefore used to quantitate both the initial rate of phagocytosis and the phagocytic capacity of human PMN. With this assay the initial rate of phagocytosis was found to be 400 μg paraffin oil-silicon oil/min/5 × 10 6 PMN, which is about 20 times higher than that reported for the same cell type using paraffin oil particles. The calculated maximum phagocytic capacity was 2.5 mg paraffin oil-silicon oil/5 × 10 6 PMN. The uptake of paraffin oil-silicon oil particles was sensitive to inhibitors of phagocytosis, such as N-ethylmaleimide, papaverine and cytochalasin B, in a dose dependent manner. The assay also permitted the detection of increased phagocytosis such as occurs in myeloperoxidase deficient PMN.