Protocol for analyzing T cell and macrophage motility in a mouse pancreatic cancer spheroid model using light-sheet microscopy

The interaction of macrophages and T cells is pivotal for shaping the immune response to cancer. Here, we present a protocol for investigating the co-culture of macrophages and T cells in a pancreatic cancer spheroid model. We describe steps for analyzing migration patterns by light-sheet microscopy, generating murine bone marrow-derived macrophages (BMDMs) and cytotoxic T lymphocytes (CTLs), and staining CTLs for image analysis. We then detail procedures for generating spheroids using a mouse pancreatic cancer cell line. For complete details on the use and execution of this protocol, please refer to Slusny et al.¹.