Protocol for isolation of live tumor-infiltrating immune cells from immunocompetent murine brains for high-dimensional profiling.

Here, we present an optimized workflow to isolate viable immune cells from tumor-bearing mouse brains, with particular emphasis on myeloid populations, to enable detailed functional analysis of the tumor-immune microenvironment. We describe steps for intracardiac perfusion and regional brain dissection. We then detail procedures for gentle enzymatic-mechanical dissociation, efficient myelin and red blood cell removal, and optional CD11b+ enrichment. This approach preserves immune cell viability and epitope diversity, supporting high-dimensional proteomic profiling via spectral flow cytometry and single-cell multi-omics.