RPMs recognize and degrade erythrocyte ghosts in vivo and in vitro. (A) Micrograph of dense erythrocytes from healthy volunteers by density centrifugation (senescent erythrocytes, left panel) and in vitro generation of ghost erythrocytes (right panel). (B-C) Senescent erythrocytes (sRBCs) and ghost erythrocytes (ghost RBCs) were stained with PKH and coincubated with a single-cell suspension of human spleen cells for 45 minutes at 37°C, were vigorously washed, and their interaction with RPMs (CD163⁺Hoechst⁺AF⁺CD14⁻ subpopulation) was quantified by flow cytometry. (D) Erythrocyte and ghost breakdown rates by RPMs were quantified by measuring mean fluorescence intensity (MFI) of PKH during a time course of 60 minutes. (E) Mice received either a transfusion of PKH- and calcein-labeled senescent erythrocytes (bottom row, red quadrant) or vehicle (top row). RPMs were gated on the basis of autofluorescence and Hoechst staining. (F-G) F4/80⁺ MACS-isolated cells from mice that received no transfusion, a fresh transfusion, or a transfusion of senescent erythrocytes were MACS isolated and subjected to benzidine staining. Attached and phagocytosed erythrocytes were quantified. Red arrows indicate phagocytosed erythrocytes and ghosts and yellow arrows indicate intact erythrocytes. Phagocytosed erythrocytes and erythrocyte ghosts (red arrows) were observed only upon transfusion of senescent erythrocytes. *P < .05; **P < .01; ***P < .001; ****P < .0001. Error bars indicate SD unless stated otherwise.