Clearance of opsonized RBCs and their uptake by F4/80-positive splenic macrophages in SHPS-1 mutant mice. (A) WT (○) or homozygous SHPS-1 mutant (•) mice at 7 weeks of age were injected with CFSE-labeled RBCs (from WT mice) that had been opsonized with IgG (right) or not (left). Clearance of the injected RBCs was determined at the indicated times thereafter as in Figure 4A. (B) Splenocytes were isolated from WT or SHPS-1 mutant (KO) mice 8 hours after transfusion of IgG-opsonized, CFSE-labeled RBCs (from WT mice) as in panel A. The splenocytes were stained with a biotin-conjugated mAb to F4/80, PE-conjugate streptavidin, and 7-AAD, after which live F4/80-positive splenic macrophages that had ingested RBCs were detected by 3-color flow cytometry. Each panel shows a density plot of all viable splenocytes; axes indicate relative fluorescence units for CFSE and PE (F4/80). Results are representative of 3 independent experiments. (C) Among all F4/80-positive cells, the percentage of cells that had ingested CFSE-labeled RBCs was determined for WT and SHPS-1 mutant recipients in experiments similar to that shown in panel B. Data are means ± SE of values from 5 mice of each group and are representative of 3 independent experiments. ^*P < .05