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 - Funding Information:
Special thanks to Fang Jin and Robin Willenbring for their help and assistance in the experiments. Also, thanks to Michael Hansen for his help and advice with the mouse work, and Dr. Kevin Pavelko for providing the TMEV-OVA virus (Mayo Clinic Immunology Department). Funding for this project was supported by NIH RO1-94168001 and NIH R21 CA186976 .
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
VL - 439
SP - 23
EP - 28
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
SN - 0022-1759
ER -