American Association of Immunologists, The Journal of Immunology, 1_Supplement(196), p. 148.14-148.14, 2016
DOI: 10.4049/jimmunol.196.supp.148.14
Elsevier, Journal of Immunological Methods, (439), p. 23-28, 2016
DOI: 10.1016/j.jim.2016.09.002
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Abstract Effective recovery of activated brain infiltrating lymphocytes is critical for research investigations involving murine neuroimmunological disease models. To optimize lymphocyte recovery, we compared isolation methods using brains harvested from 7 day Theiler’s virus infected mice. Brains were processed using either a mechanical dounce based approach or were enzymatically digested 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 CD44hiCD62loactivated lymphocytes using dounce method. Also, a 3-fold greater recovery of activated virus specific CD8 T cells for the immunodominant virus epitopes Db: VP2121-130 and Kb: OVA257-264 was achieved through mechanical dounce homogenization approach as compared to collagenase digest. Greater percentage of viable cells was also achieved with the dounce homogenization. We therefore conclude that manual homogenization is a superior approach to isolate activated T cells from the mouse brain.