Phagocytosis of RBCs by neutrophils induces a strongly diminished respiratory burst. (A) Release of H₂O₂ was measured by Amplex Red assay for neutrophils incubated ± RBC or RBC-ops. No burst was measured in the 3 conditions (mean ± SEM; n = 10-12). To control for the ability to produce a respiratory burst, neutrophils from all 3 conditions were stimulated with zymosan or PMA (mean ± SEM; n = 10-12). (B) Release of intracellular H₂O₂ production was measured using a DHR assay with neutrophils incubated ± RBC or RBC-ops. Neutrophils incubated with zymosan served as a positive control. Addition of PMA at 180 minutes showed neutrophils in all conditions were capable of producing ROS (mean ± SEM; n = 4-13). Asterisks above data points represent significant differences for zymosan compared with control (**P < .01; *P < .05). (C) Respiratory burst was visualized using NBT, which produces a blue precipitate in presence of intracellular ROS. Neutrophils were incubated plus RBC or RBC-ops. Neutrophils plus PMA served as a positive control and neutrophils alone served as a negative control. Images are representative for 3 individual experiments. May-Grünwald Giemsa stain; original magnification ×500. RFU, relative fluorescence unit.