Distribution of CR1 on erythrocytes depends on the staining method. (A) Erythrocytes from a HH donor were incubated with anti-CR1 mAb 1F11, followed by AlexaFluor-594 goat anti–mouse Ab. After each step the cells were centrifuged, dispersed, and resuspended in fresh buffer. The majority of CR1 was in large clusters. (B) Fresh erythrocytes from the same HH donor were fixed as described in “Fresh and fixed erythrocytes” and incubated with anti-CR1 mAb 1F11 followed by AlexaFluor-594 goat anti–mouse Ab. CR1 is seen as small speckles throughout the plasma membrane. Bar represents 8 μm. (C) Erythrocytes processed for panel A were imaged within 1 minute after the addition of secondary antibody without any washing. Although larger CR1 clusters can already be observed (white arrows), most of the CR1 is still seen as small speckles. The high background of the image is due to the presence of the secondary antibody in solution during imaging. Bar represents 8 μm. (D) Erythrocytes were incubated with AlexaFluor-594–labeled Fab anti–CR1 mAb 2B11 for 15 minutes at room temperature, washed once, and resuspended in fresh buffer. The majority of CR1 is dispersed with few larger clusters. (E,F) Fresh unfixed (E) or fixed (F) erythrocytes were incubated with C3b-beads AlexaFluor-488 (open histogram) or control beads (IgG + heat-inactivated human serum, filled histogram) for 30 minutes at room temperature. Erythrocytes were then washed and analyzed by flow cytometry. Net mean fluorescence intensity (MFI) was 5.38 for fresh erythrocytes and 3.01 for fixed erythrocytes. (G) On fresh erythrocytes, CR1 partitions in the cytoskeletal fraction. Cytoskeletal (C) and membrane (M) fractions isolated as described in “Immunoblotting and coimmunoprecipitation” were probed for CR1 using rabbit polyclonal IgG. Whole erythrocyte lysate (E, lane 1; 1.5 × 106 erythrocytes/lane) was used as positive control.
