A Quantitative Assessment of the Phagocytosis of Allogeneic and Xenogeneic Erythrocytes by Rat Macrophages <em>In Vitro</em>

Xenogeneic cell transplantation often faces significant immune rejection, even in immunodeficient animal models. Among residual immune components, macrophages can actively phagocytose transplanted human cells, posing a challenge to long-term engraftment. To address this, we developed a standardized in vitro assay to quantify macrophage-mediated phagocytosis of human versus rat red blood cells (RBCs). Rat peritoneal macrophages were isolated, cultured, and exposed to either human or rat RBCs. Human RBCs were identified using CD235a, while rat RBCs were pre-labeled with DeepRed. Flow cytometry analysis was conducted using CD163 to identify macrophages and propidium iodine (PI) exclusion to gate living cells. The results showed a substantial difference in phagocytic activity: 5.08% of macrophages engulfed human RBCs, while only 1.59% phagocytosed rat RBCs, yielding a relative phagocytosis index of 3.21. This protocol allows for quantitative assessment of immune compatibility and offers a reproducible method to evaluate innate immune responses in xenotransplantation. It holds potential for refining donor-recipient selection and guiding immunosuppressive strategies in translational research.