Figure 2.

Consumption of red blood cells (RBCs) by different phagocytic subsets. RBCs were labeled with DiO⁺ and transfused into the indicated recipient strains. Subsets of recipient splenocytes were enumerated by flow cytometry using standard staining panels for lineage specific antigens (A,B). The extent of RBC consumption was assessed by gating on each indicated lineage and assessing DiO fluorescence, which reflects the extent of RBC consumption (8) (C,D). Only events that stained negative with anti-TER119 (an RBC specific antigen in mice) were counted, so as to avoid evaluating phagocytes with RBCs stuck to the surface, as opposed to consumed. Representative data are shown for one experiment with three mice per group. Similar results were obtained in three out of three repetitions. For cellular subset analysis, T cells, B cells, and RBCs were excluded from total live leukocytes by gating on Thy1.2⁻CD19⁻TER119⁻ cells. Red pulp macrophages (RPM) are defined as CD11c^intF480⁺CD11b^lo/−, CD8a⁻ dendritic cells (DCs) express CD11c^hiCD11b⁺CD8a⁻ whereas CD8a⁺ DCs are CD11c^hiCD11b⁻CD8a⁺, and plasmacytoid DCs (pDCs) are CD11c^intLy6C^hiLLy6G⁻B220⁺. CD11c^int/−CD11b⁺Ly6G⁻Ly6C^hiCD115⁺ inflammatory monocytes, CD11c^int/−CD11b⁺Ly6G⁻Ly6C^hiCD115⁻ resident monocytes, CD11c^int/−CD11b⁺Ly6G⁺ neutrophils, and CD11c^int/−CD11b⁺Ly6G⁻ with high side scatter eosinophils were also be delineated.