Protocol for in vivo immune cell analysis in subcutaneous murine tumor models using advanced flow cytometry.

Here, we present a protocol for guiding tissue preparation and flow cytometric analysis in subcutaneous murine tumor models and secondary lymphoid organs. We describe steps for dissociating tumors, spleens, and lymph nodes to obtain single-cell suspensions. We then detail procedures for immune cell staining and analysis and gating strategies including the use of fluorescence-minus-one controls (FMOs). This approach provides valuable insights into the impact of cancer therapies on the tumor and systemic immune response. For complete details on the use and execution of this protocol, please refer to Ingelshed et al.1.