In vitro phagocytosis assay and anti-CD47 antibody treatment

CFSE-labeled target cells (10⁵) were cocultured with RAW 264.7 murine macrophage cells (5 × 10⁵), differentiated human U937 cells (5 × 10⁵), or DC98 dendritic cells (5 × 10⁵) for 1 h at 37°C in a humidified atmosphere containing 5% CO2. Cells were stained with CD11b or CD11 c and were subjected to flow cytometric analysis. To block CD47, CFSE-labeled target cells (10⁶ cells/mL) were treated with an anti-mouse CD47 antibody (LEAF^TM purified anti-mouse CD47 antibody, Biolegend, CA) for 20 min at 37ºC. Cells were washed, resuspended in culture medium, and cocultured with macrophages and DCs.