A protocol for high-throughput screening for small chemicals promoting macrophage-mediated tumor cell phagocytosis in mice

Macrophage-mediated phagocytosis has emerged as a pivotal mechanism for eliminating tumor cells within the realm of cancer immunotherapy. Here, we present a protocol for identifying small molecules that enhance phagocytosis in mice using a co-culture system comprising primary macrophages, cancer cells, and a blockade of phagocytic checkpoints. We describe steps for expressing enhanced green fluorescent protein-luciferase (eGFP-Luc) and producing bone marrow-derived macrophages (BMDMs). We then detail procedures for optimizing co-culture conditions for high-throughput screen (HTS) and executing HTS chemical identification. For complete details on the use and execution of this protocol, please refer to Cao et al. ¹.