Development of a flow cytometry panel for microglia and immune cell populations in a brain tumor model

Accurate characterization of microglia and infiltrating immune cell populations is essential for understanding the immunological landscape of brain tumors and for evaluating therapeutic responses. Here, we report the development and optimization of a multicolor flow cytometry panel tailored to discriminate resident microglia from peripheral myeloid and lymphoid subsets within an experimental brain tumor model. Marker selection was guided by tissue-specific expression profiles, leveraging differential levels of CD45, CX3CR1, and P2RY12 to resolve microglia, while incorporating canonical markers for infiltrating macrophages, dendritic cells, neutrophils, and T cells. Enzymatic dissociation conditions, viability dye selection, and gating hierarchy were systematically optimized to preserve epitope integrity and maximize signal resolution. Validation using tumor-bearing and control brain tissues demonstrated robust separation of microglial and infiltrating populations with reproducible quantification across biological replicates. This workflow provides a reliable and scalable approach for immune profiling in brain tumor research, enabling deeper insight into neuroimmune interactions and immunotherapy-driven changes within the tumor microenvironment.