The native and engineered RBCs were fixed with 4% glutaraldehyde at 4°C overnight and washed twice with a 10 mM PBS buffer solution. The specimens were incubated with commercial anti-D (Rh) monoclonal antibodies (IgG; dilution, 1:10; 4A Biotech, China) or anti-B monoclonal antibodies (IgM; dilution, 1:10; ab24224, Abcam) for 1 hour at room temperature. Subsequently, the samples were washed three times with PBS and incubated with Alexa Fluor 568 goat anti-mouse IgG fluorescent dye (dilution, 1:200; ab175473, Abcam) for 2 hours at room temperature. The stained cells were analyzed using an FC500 MPL flow cytometer (Beckman Coulter, CA) and FXP software (CXP 2.1).

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