Interaction between macrophages and AMIS-inducing antibody sensitized RBCs triggers erythrocyte membrane nibbling by macrophages in vitro. (A) RAW macrophages were seeded on coverslips for 2 days and maintained in complete RPMI at 37°C and 5% CO₂. PKH67⁺HOD-RBCs were opsonized with pIgG anti–HEL, anti-Duffy CBC-512 IgG2a, or kept nonsensitized (PBS) before incubation with macrophages at 37°C and 5% CO₂. After 3 hours, excess RBCs were washed away, and external RBCs lysed followed by fixation and imaging on a Quorum multimodal imaging system (spinning disk confocal) under original magnification ×63 objective oil immersion (numerical aperture 1.47). Scale bar = 10 μm. (B) RAW macrophages were seeded on coverslips and, 1 day later, were transfected with a plasmid expressing the F-actin visualizing protein LifeAct monomeric red fluorescent protein. Twenty-four hours later, transfected macrophages were transferred to a chamlide chamber for confocal microscopy and PKH67⁺HOD-RBCs sensitized with anti-Duffy MIMA29 were added to the macrophages 5 minutes before imaging to allow the RBCs to reach the bottom. For areas where RBCs and macrophages appear to be in proximity, images were taken every 60 seconds for 45 minutes on a spinning disk confocal as above. Scale bar = 5 μm. The white square at the 9 minutes time point indicates the zoomed area (bottom 2 rows). Zoom: scale bar represents 2 μm. There was a higher apparent level of trogocytosis of RBC membrane fragments by macrophages using the confocal imaging with fixed samples (A) as compared with the LifeAct experiments (B). This is likely explained by the shorter incubation time for the LifeAct experiments (45 minutes vs 3 hours) and phototoxic effects of the repeated imaging for live-cell experiments.