Hemophagocytosis is responsible for the anemia induced by BA treatment. Flow cytometry analysis was performed to identify and measure the interaction between macrophages and RBCs, an indicator of hemophagocytosis. The figure shows the results obtained in the spleen of 2 representative mice, treated with PBS and BA, respectively, and analyzed 2 days after BA injection. Three mice per group were analyzed, obtaining similar results. Gating steps were performed as indicated from A to E. Briefly, (A) we first excluded the Gr1⁺/CD115⁻ and Gr1⁺/CD115⁺ populations (neutrophils and Gr1^hi monocytes) and gated both the Gr1⁻/CD115⁻ and Gr1⁻/CD115⁺ cells. (B) These were further divided into an F4/80^hi/CD115⁻ population that also included eosinophils. (C) Eosinophils were excluded by their side scatter (SSC)^hi distinctive feature. (D) The remaining F4/80^hi population, comprised of mature macrophages, was analyzed by plotting SSC-H vs SSC-A, which allows discriminating multiplets vs single cells. Macrophages/RBC multiplets (indicating macrophages binding RBCs during the hemophagocytosis process) were discriminated using F4/80 and Ter119. (E) The increase of Ter119-positive cells (RBCs) among the multiplets indicated that hemophagocytosis was increased in BA-treated mice compared with PBS controls.