Characterization of murine lymphocyte activation and exhaustion markers by a 14-color flow cytometry panel

Previously designed flow cytometry panels have provided a framework to analyze T-cell activation; however, few provide an extensive view of lymphocyte populations, and none are optimized for murine models. This article describes a panel designed specifically to assess the expression of activation and exhaustion markers in expanding lymphocyte populations in tumor-bearing mice across two distinct genetic backgrounds: BALB/c and C57BL/6. This comprehensive panel enables the assessment of multiple functional states and immune checkpoint markers across cytotoxic CD8+ T cells, helper and regulatory CD4+ T cells and natural killer cells in murine whole blood, lymph nodes and tumor. Plain language summary Flow cytometry is a technique that allows researchers to analyze protein expression on single cells through the detection of fluorescence markers that is widely used to assess immune cell phenotypes. The selection of target proteins expressed on specific cell subsets and accompanying fluorophores is known as ‘panel building' and is critically important for accurate flow cytometry results. However, there is a lack of optimized and reproducible panels for mouse models of cancer. This article describes the development and performance of a robust panel that characterizes immune cell diversity and activation, while leaving room for customization. This flow cytometry panel provides a starting point for exploring the spectrum of mouse lymphocyte activation and is adaptable for subsequent studies.