Concurrent measurement of antigen- and antibody-dependent oxidative burst and phagocytosis in monocytes and neutrophils

The current study aims to review flow cytometric (FCM) parameters for the q uantification of phagocytosis. A limitation of existing methods is their di fficulty with accurate quantification of the phagocytic index, i.e., number of beads per phagocyte, in individual cell lines in mixed cell suspensions . We have quantified phagocytosis and the oxidative burst simultaneously us ing fluorescent beads coated with meningococcal outer membrane vesicles (OM V beads) by the conversion of dihydrorhodamine 123 (DHR-123) to rhodamine 1 23 (R-123). Both these processes depend on specific serum opsonins. After t he incubation, staining with a fluorescent anti-CD14 monoclonal antibody su cceeded in discriminating phagocytosing monocytes from neutrophils. The spe ctral overlaps between OMV beads, R-123, and anti-CD14 could be completely compensated. Percentage of phagocytosis and the phagocytic index were simil ar in monocytes and neutrophils, but the oxidative burst behaved differentl y. Two monocyte subpopulations were observed. Both subpopulations spontaneo usly converted some DHR-123 into R-123, whereas the reaction was triggered by phagocytosis in neutrophils. The total oxidative response increased with increasing phagocytic index in both cell types, but the oxidative burst in monocytes was about twice that of neutrophils. The oxidative ratio (mean R -123 fluorescence value divided by the phagocytic index) declined with time in monocytes, but increased in neutrophils. Our results demonstrate the ne ed for careful attention to technical details. This single-laser, three-col or FCM method facilitates the comparative research of phagocytosis and the oxidative burst in monocytes and neutrophils and provides a basis for a num ber of applications in hematology, infectious medicine, and immunology. (C) 2000 Academic Press.