Characterization of phagocytosis of P-RBC complexes. (A) Mice were transfused with PKH26-labeled (donor) RBCs and platelet-labeling antibody (anti-GPIbβ-DyLight649, X649). Single-cell liver and spleen preparations harvested 2 or 20 hp were stained and analyzed by flow cytometry using the method depicted. Phagocytes were defined as CD45⁺, CD3ε^–, CD19^–, and NK1.1^– (Lin^–); CD11b^lo and F4/80^hi and subsequently classified as those containing only donor RBCs (PKH26⁺, X649^–), only platelets (PKH26⁻, X649⁺), or both RBCs and platelets (PKH26⁺, X649⁺). Bottom panels show proportions of the different phagocyte types in spleens from nontransfused mice and those transfused with only donor RBC or only X649 (as controls) or both RBC and X649. (B) Proportions of phagocytes in spleen and liver containing donor RBCs, platelets, or both RBCs and platelets; 5 mice and 1 experiment. (C) Fluorescence microscopy images of spleen collected 20 hp showing macrophages (F4/80-stained) containing donor-derived RBCs (PKH26-labeled, arrowheads) and both donor RBCs and X649-labeled platelets (arrows); Scale bar, 10 μm. Additional images from other mice shown in supplemental Figure 6. (D-G) Cohorts of mice were pretreated 24 hours prior with platelet-depleting (anti-GPIbα or anti-αIIbβ3) or IgG isotype (control) antibodies and along with Mpl^−/− and Mpl^+/+ mice, transfused with PKH26-labeled donor RBCs (D) and analyzed after 20 hours for proportions of splenic (E) and liver (F) erythrophagocytes. (G) Blood samples collected at take down were analyzed for PKH26-labeled RBC levels in bloodstream using the gating strategy depicted. Singlet RBC were identified by anti-ter119 staining. A nontransfused mouse (nil) is shown for comparison; 5 to 8 mice per group, 3 independent experiments. Symbols and horizontal bars indicate individual mice and means, respectively. Comparisons shown using ANOVA. IgG, immunoglobulin G.