Isolation of mouse mammary carcinoma-derived macrophages and cancer cells for co-culture assays.

We recently established an in vitro co-culture system in which monophosphoryl lipid A + interferon-γ (MPLA+IFNγ)-treated tumor-associated macrophages (TAMs) killed cancer cells. Here, we describe a step-by-step protocol for isolating TAMs and cancer cells from mouse primary mammary carcinomas, the setup of the co-culture system, and the image acquisition approach. The technical difficulties in the co-culture assay involve isolating pure TAMs and cancer cells from the same tumor and staining them with different dyes to track the macrophages' tumoricidal activity. For complete details on the use and execution of this protocol, please refer to Sun et al. (2021).1.