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J Immunol Methods. 2016 Dec;439:23-28. doi: 10.1016/j.jim.2016.09.002. Epub 2016 Sep 10.

Superior isolation of antigen-specific brain infiltrating T cells using manual homogenization technique.

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Immunology Graduate Program, Mayo Clinic, Rochester, MN, United States.
Department of Immunology, Mayo Clinic, Rochester, MN, United States. Electronic address:


Effective recovery of activated brain infiltrating lymphocytes is critical for investigations involving murine neurological disease models. To optimize lymphocyte recovery, we compared two isolation methods using brains harvested from seven-day Theiler's murine encephalomyelitis virus (TMEV) and TMEV-OVA infected mice. Brains were processed using either a manual dounce based approach or enzymatic digestion using type IV collagenase. The resulting cell suspensions from these two techniques were transferred to a percoll gradient, centrifuged, and lymphocytes were recovered. Flow cytometric analysis of CD45hi cells showed greater percentage of CD44hiCD62lo activated lymphocytes and CD19+ B cells using the dounce method. In addition, we achieved a 3-fold greater recovery of activated virus-specific CD8 T cells specific for the immunodominant Db:VP2121-130 and engineered Kb:OVA257-264 epitopes through manual dounce homogenization approach as compared to collagenase digest. A greater percentage of viable cells was also achieved through dounce homogenization. Therefore, we conclude that manual homogenization is a superior approach to isolate activated T cells from the mouse brain.


CD8 T cell; CNS viral infection; Lymphocyte isolation; Neuroinflammation; Optimized recovery; Theiler's murine encephalomyelitis virus

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