Measurement of phagocytosis utilizing [14C]cyanate-labelled human red cells and monocytes

Human red cells were labelled with [¹⁴C]cyanate, non-oxidant, permeant reagent that binds irreversibly to amino groups in proteins. Cyanate did not modify GSH levels, nor glucose-6-phosphate dehydrogenase (G6PD) activity when added at the labelling concentration i.e. c. 0.5 mM. Phagocytes were human monocytes, isolated and plated on 16 mm diameter plastic wells. Each well was plated with 40-70x10³ cells. Monocytes were quantified by DNA assay with the DNA intercalating fluorescent compound Hoechst 33258. The basal phagocytic rate of normal red cells by monocytes was 0.34±0.21 red cell per monocyte. Treatment of normal red cells with 20 μM diamide, or 50-100 μM chromate enhanced this rate 10-15-fold. 10μM diamide or chromate were sufficient to stimulate phagocytosis of G6PD-deficient (Mediterranean variant) red cells.