TY - JOUR
T1 - Superior isolation of antigen-specific brain infiltrating T cells using manual homogenization technique
AU - Cumba Garcia, Luz M.
AU - Huseby Kelcher, April M.
AU - Malo, Courtney S.
AU - Johnson, Aaron J.
N1 - Publisher Copyright:
© 2016 Elsevier B.V.
PY - 2016/12/1
Y1 - 2016/12/1
N2 - 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.
AB - 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.
KW - CD8 T cell
KW - CNS viral infection
KW - Lymphocyte isolation
KW - Neuroinflammation
KW - Optimized recovery
KW - Theiler's murine encephalomyelitis virus
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U2 - 10.1016/j.jim.2016.09.002
DO - 10.1016/j.jim.2016.09.002
M3 - Article
C2 - 27623324
AN - SCOPUS:84997818372
SN - 0022-1759
VL - 439
SP - 23
EP - 28
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
ER -