Fig. 1.

Designs and characterization of engineered RBCs. (A) Schematic for Kell C-terminal sortase labeling with GGG-carrying antigens peptides. (B) Evaluation and quantification of mature Kell-LPETGG RBCs for sortase labeling by incubation of RBCs with biotin-containing probes in the presence or absence Sortase A. Cytofluorimetry was performed with anti-TER119, a RBC surface marker, and antibiotin antibodies. (n = 3; **P < 0.01, unpaired t test with Holm–Sidak adjustment). (C) Quantification of sortase labeling of Kell-LPETGG RBCs with different biotin-containing peptides by immunoblotting (IB) using streptavidin-HRP. GFP-biotin carrying a single biotin/mole of GFP was used a reference. (D) CFSE-labeled RBCs from C57BL/6J and Kell-LPETGG mice were transfused into recipient mice. Kell-LPETGG blood samples were also subjected to sortagging with the three different OVA-derived peptides before transfusion. RBC survival in the circulation was tracked via CFSE fluorescence by flow cytometry.