CD47 inhibits FcγR-mediated phagocytosis of untreated RBCs, but not serum-dependent phagocytosis of Ox-RBCs. (A) WT or CD47^−/− RBCs were opsonized with 1, 5, or 25 μg/mL mAb 34-3C, washed, and then incubated with RPM for 30 minutes at 37°C. Data are mean plus or minus SEM for 4 coverslips in one representative experiment of 3. (B) Following oxidation with Cu/ascorbate for 60 minutes, WT Ox-RBCs and CD47^−/− Ox-RBCs (gray histograms) bound similar levels of FITC-conjugated annexin V, as determined by flow cytometry (dotted line: RBCs incubated in PBS). (C) WT or CD47^−/− Ox-RBCs were incubated with adherent BMM or RPM for 60 minutes at 37°C in the presence of serum. Phagocytosis was evaluated as described in the legend to Figure 1. Data represent mean plus or minus SEM for 3 to 6 individual experiments.