Investigating CD8 T Cell Exhaustion in Chronic LCMV Infection by Flow Cytometry

Abstract CD8 T cells and the process of T cell exhaustion are central topics in both basic immunology and translational research, including clinical applications. Here, we provide a comprehensive, step‐by‐step guide to investigate CD8 T cells in murine in vivo lymphocytic choriomeningitis virus (LCMV) infection models. We outline viral stock preparation, infection, and viral titer evaluation. We put an emphasis on flow cytometry as a method to investigate phenotypical and functional characteristics of CD8 T cell exhaustion, but the protocols provided will also facilitate imaging or transcriptomic analyses. With these resources, we aim to streamline establishment of the LCMV model for new users and to support harmonization and optimization of workflows for laboratories already working with this model. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Virus production Basic Protocol 2 : Virus titer quantification from virus stocks by focus‐forming assay Alternate Protocol 1 : Virus titer quantification from infected tissues by focus‐forming assay Basic Protocol 3 : Peptide‐MHC‐I tetramer production Basic Protocol 4 : Naïve CD8 T cell isolation using StemCell TM kit Alternate Protocol 2 : Naïve CD8 T cell enrichment using MojoSort TM kit Basic Protocol 5 : Adoptive CD8 T cell transfer and LCMV infection Basic Protocol 6 : Animal husbandry Basic Protocol 7 : Preparation of single‐cell suspension from secondary lymphoid organs Alternate Protocol 3 : Preparation of single‐cell suspension from peripheral tissues Basic Protocol 8 : Extracellular and intracellular staining Basic Protocol 9 : Restimulation analysis Basic Protocol 10 : Sorting