RPMs are the predominant splenic population responsible for phagocytosis of old RBCs, with Ly6C^himonocytes also contributing to RBC clearance. Wild-type C57BL/6 mice were transfused with 200 μL of PBS, fresh GFP⁺ RBCs, or old GFP⁺ RBCs. Mice were then sacrificed at 2 or 24 hours posttransfusion. (A) Representative dot plots show the gating strategy for RPMs (F4/80^hi, VCAM-1⁺, CD11b^lo), Ly6C^hi monocytes (Ly6C^hi, CD11b^hi), granulocytes (Ly6G⁺, Ly6C⁺, CD11b⁺), CD8⁺ DCs (CD8⁺, CD11b⁻, MHCII^hi, CD11c^hi), and CD11b⁺ DCs (CD8⁻, CD11b⁺, MHCII^hi, CD11c^hi) at 2 hours posttransfusion. These cells were obtained from the spleen at 2 hours posttransfusion. Percentages of GFP⁺ cells are depicted; these represent nucleated cells that have phagocytosed GFP-expressing fresh or old transfused RBCs. (B) Frequency of GFP⁺ RBC phagocytosis at 2 and (C) 24 hours posttransfusion; by 24 hours posttransfusion, the levels of phagocytosis were dramatically reduced compared with the 2-hour time point and the previously ingested GFP⁺ RBCs were degraded. (D) Hmox-1 (ie, HO-1) expression by RPMs 5 hours posttransfusion. (E) Spic expression by Ly6C^hi monocytes 5 hours posttransfusion. Data representative of 4 experiments with at least 3 mice per group; *P < .05; ***P < .001; ****P < .0001; ANOVA with Tukey or Sidak multiple comparison test.