Shape of rigid RBCs modulates CD47’s “don’t eat me” signal. (A) A flow cytometry-based phagocytosis assay shows that macrophage phagocytosis of high antiserum opsonized native RBCs, GA stomatocytes, and GA discocytes is significantly higher than unopsonized cells (*P < .05 compares indicated conditions; ** P < .05 compares high antiserum to zero antiserum for each RBC condition: native RBC, GA stomatocytes, and GA discocytes). (B) RBCs treated with a mild hypotonic buffer followed by GA reaction generated rigid, more rounded, GA stomatocytes (upper inset image). A flow cytometry-based phagocytosis assay of high antiserum opsonized GA stomatocytes shows less engulfment than GA discocytes in the absence of anti-CD47 blocking antibody, whereas blocking CD47 equalized uptake (*P < .05; n ≥ 5000 macrophage in duplicate, ±SD). Time-lapse imaging of the macrophage phagocytic synapse with the stomatocyte shows pseudopods closely surrounding the rounded cell (lower inset image).