Humanized Mouse Model Establishment

Prior to injection, blood was stored at 4°C and washed 2x with RPMI 1640, 25 mM HEPES (Sigma-Aldrich, St. Louis, MO) containing 7x10^-3 mM hypoxanthine (Sigma-Aldrich, St. Louis, MO) at RT. The buffy coat was removed by aspiration with erythrocytes resuspended at 50% hematocrit in RPMI 1640, 25% decomplemented human serum (Sigma-Aldrich, St. Louis, MO), and 3.1 mM hypoxanthine. huRBC suspension was warmed at 37°C for 20 min prior to intraperitoneal (i.p.) injection. Mice were i.p injected with 1 ml of huRBCs containing 70% O⁺ human erythrocytes in RPMI 1640 (25 mM HEPES, 2 mM L-glutamine) supplemented with 50 μM hypoxanthine plus 10% human serum. Engraftment continued daily using 1 ml huRBCs throughout the experiment, in order to reach the minimum desired percentage of huRBCs ≥ 70% (measured by flow cytometry as previously described [33 (link)]) by 7 days post mosquito bite challenge.