![]() ![]() ![]() High dimensional data increasingly requires the help of dimensionality reduction and unbiased analysis techniques therefore, we developed a protocol to utilize the FlowJo-based plugins to perform an unbiased cell classification for validation of our manual gating strategy. Furthermore, we titrated these antibodies in multiple organs to maximize their staining index. We optimized antigen-fluorophore combinations to minimize background and overlap, as shown by a spillover-spreading matrix. Lineage antigens were identified through extensive literature mining, and clones were chosen based on the highest number of citations from a publicly available database. Here, we developed an intracranial tumor processing protocol and a 14-color panel to resolve 13 distinct immune cell populations present in murine models of GBM. Flow cytometry provides the best means of accomplishing this objective, since it boasts high, single cell resolution and accessibility, with an ever-growing number of compatible antibodies. For these reasons, it is critical to have a reliable and reproducible way of identifying all the major immune populations present in the tumor microenvironment. Additionally, it is now accepted that subtle functional and phenotypic changes to these populations can predict response to treatment. GBM has a complex TME composed of many lymphoid and myeloid populations, each regulating tumor progression. Recent failures in immunotherapeutic strategies for the treatment of GBM have shifted the focus towards understanding the immune populations in the tumor microenvironment (TME) to better identify and target barriers to success. Despite extensive medical intervention and therapeutic advancements in other solid tumors, the median survival remains 15–21 months. Glioblastoma (GBM) is the most common primary brain tumor in adults. ![]()
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