mHEL RBCs that have been transfused into HEL/CFA-immunized mice have a normal circulatory life span and morphology despite loss of antigen. A mixture of leukoreduced CM-DiI–labeled mHEL RBCs and DiO-labeled C57BL/6 RBCs were transfused into HEL/CFA-immunized mice. Antigen loss was confirmed by staining with anti–mouse Ig or anti-HEL followed by anti–mouse Ig (data not shown). At the indicated time points, the percentage of remaining transfused mHEL or C57BL/6 RBCs was determined by flow cytometry (A). After confirmation of mHEL antigen loss, mHEL RBCs (CM-DiI) and C57BL/6 RBCs (DiO) were reisolated by flow-activated cell sorting (FACS) and analyzed by peripheral blood smear (B). The experiments shown in this figure have been reproduced in at least 3 separate experiments. The data presented in this figure are representative results. Flow sorting for morphologic analysis was performed twice. Slides were visualized using a Nikon Eclipse E400 microscope (Nikon, Melville, NY) with a 10× ocular and either 40×/0.65 NA or 100×/1.25 NA oil lenses. Photographs were taken using a Spot Insight color digital camera, and images were acquired using Spot Advanced software, version 4.0.9 (Diagnostic Instruments, Sterling Heights, MI). Pictures were cropped and brightness and contrast were adjusted using Adobe Photoshop (Adobe Systems, San Jose, CA).