Supplementary Figure 1.: Validation of flow cytometry–based in vitro phagocytosis assay

(a) Representative flow cytometry plots of in vitro phagocytosis assay. Macrophages are defined as CD45⁺ events, and tumor cells as GFP⁺ events. CD45⁺, GFP⁺ events correspond to macrophages that have phagocytosed target cells. (b) Representative images from an automated microscopy-based approach for quantifying in vitro phagocytosis. Using a custom program on the GE Cytell, we defined human macrophages based on staining with anti-CD45-PE antibody (yellow), and tumor cells based on GFP expression (green). Phagocytic events are defined based on overlap of these two signals (right panel, white arrowheads). (c) Comparison of FACS-based quantification (left) or automated microscopy-based quantification (right) of DLD1 phagocytosis under conditions of PBS treatment, treatment with the anti-CD47 antibody Hu5F9-G4, or treatment with the anti-EGFR antibody Cetuximab.