DiO fluorescence is not due to nonspecific transfer of DiO, nor to RBC adherence to the APC surface. Twenty-four hours after transfusion with DiO-labeled syngeneic RBCs, splenocytes were harvested, and nonphagocytic T cells were visualized by staining with anti-CD3 (A). Specificity was confirmed by staining with an isotype-matched control (B). After gating on CD3⁺ T cells, DiO fluorescence was measured both in mice transfused with DiO-labeled RBCs (–) or untransfused negative control mice (------) (C). In a separate control experiment, splenic macrophages were visualized by anti-F4/80 staining (D). After gating on the F4/80⁺ population, fluorescence of anti-TER-119 was analyzed (E). RBCs were stained with anti-TER-119 as positive control for antibody activity (F). Staining from representative mice is shown, and y-axes of histograms represent percentage of maximum peak value. This experiment was performed 3 times with similar results. For panels A, B, D, E, numbers represent percentage of cells in gate.