The oxygen affinity and Hill plots of the native and engineered RBCs were calculated from an oxygen equilibrium curve measured with a Hemox analyzer (TCS Scientific, CA) at 37°C. The samples were diluted with PBS (pH 7.2, 37°C). The 2,3-DPG content in the RBCs before and after the framework modifications was quantified using a 2,3-DPG test kit (Sigma). The ATP content was also investigated after framework engineering using the ENLITEN ATP Assay System (Promega, CA). The UV-Vis absorption spectra were measured using a T6 UV-Vis spectrometer (Puxi, China). The Lorrca system (Mechatronics) was used to determine all the aggregation measurements of native RBCs, engineered RBCs, and mixtures of native and engineered RBCs by syllectometry. Human umbilical vein endothelial cells (HUVECs) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, maintained at 37°C in humidified air and 5% CO2, and used for the flow adhesion experiments. The HUVECs were cultured in a flow chamber (ibidi, Germany), examined using an Eclipse TE2000-S microscope (Nikon, Japan), and perfused with native RBCs, engineered RBCs, and mixtures of native and engineered RBCs at a shear rate of 1.5 dynes/cm for 20 min and then with cell-free solution for rinsing at the same shear rate. The assays of clot dynamics parameters (R, K, α, and MA) were performed using the TEG test (TCA 6000, China).

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