Protocol for multimodal programming of murine macrophage phenotypes

Macrophages continuously sense their environment and adopt distinct functional states in response to danger, repair, and homeostatic signals. Here, we present a protocol for generating murine bone marrow-derived macrophages and programming them with microbial/TLR and nucleic acid-sensing agonists, IL-4, apoptotic cells, and tumor microenvironment analogs. We describe steps for stimulation conditions and downstream immunological assays, including flow cytometry, ELISA, RT-PCR, and multiomics, to comprehensively profile murine macrophage phenotypes and functions across these contexts. For complete details on the use and execution of this protocol, please refer to Mirji et al.¹; Mirji et al.²; and Shinde et al.³.