Time-Lapse Imaging of Antibody-Driven Macrophage Phagocytosis of Green Fluorescence Protein-Labeled Triple-Negative Breast Cancer Cells.

Antibody-dependent cellular phagocytosis is a key mechanism by which macrophages engulf antibody-opsonized cancer cells. This protocol describes a time-lapse live-cell imaging method to visualize antibody-dependent macrophage phagocytosis of green fluorescent protein-labeled, CD24-expressing triple-negative breast cancer cells. Macrophages are derived from human peripheral blood mononuclear cells, differentiated with macrophage colony-stimulating factor, and polarized using interferon gamma and lipopolysaccharide to generate a pro-inflammatory phenotype. Cancer cells are opsonized with an anti-CD24 monoclonal antibody prior to co-culture with macrophages at a defined effector-to-target ratio. Phagocytic events are recorded over a 16 h period using an automated microscope equipped with environmental control to maintain physiological conditions. Imaging is performed using brightfield and fluorescence channels, allowing identification of cancer cells and monitoring of their internalization by macrophages. Representative time-lapse images demonstrate the progressive engulfment of fluorescent cancer cells over time. To enable quantitative assessment, a parallel endpoint assay is performed under identical co-culture conditions, in which non-internalized cancer cells are removed prior to imaging to allow accurate measurement of phagocytic events. This method provides a standardized and reproducible approach for visualizing antibody-driven macrophage phagocytosis in vitro. Importantly, the integration of time-lapse imaging with endpoint-based quantification enables complementary evaluation of the kinetics and magnitude of phagocytosis. This protocol supports the evaluation of therapeutic antibodies and macrophage-directed cancer immunotherapies in a controlled in vitro setting.