Superior isolation of antigen-specific brain infiltrating T cells using manual homogenization technique.
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 CD45hicells showed greater percentage of CD44hiCD62loactivated 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:VP2121-130and engineered Kb:OVA257-264epitopes 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.
Immunology Graduate Program, Mayo Clinic, Rochester, MN, United States.
Luz M
LM
Cumba Garcia
Immunology Graduate Program, Mayo Clinic, Rochester, MN, United States.
April M
AM
Huseby Kelcher
Immunology Graduate Program, Mayo Clinic, Rochester, MN, United States.
Courtney S
CS
Malo
Department of Immunology, Mayo Clinic, Rochester, MN, United States. Electronic address: Johnson.Aaron2@mayo.edu.
Aaron J
AJ
Johnson
D000818:Animals; D000941:Antigens / Q000276:immunology*; D018941:Antigens, CD19 / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology; D001921:Brain / Q000276:immunology* / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D036022:Capsid Proteins / Q000276:immunology; D018188:Cardiovirus Infections / Q000276:immunology* / Q000821:virology; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002499:Centrifugation, Density Gradient; D024141:Collagen Type IV / Q000378:metabolism; D017364:Collagenases / Q000378:metabolism; D004195:Disease Models, Animal; D000939:Epitopes; D005260:Female; D005434:Flow Cytometry; D018960:Hyaluronan Receptors / Q000276:immunology; D008213:Lymphocyte Activation; D008297:Male; D008810:Mice, Inbred C57BL; D010047:Ovalbumin / Q000276:immunology*; D011205:Povidone / Q000737:chemistry; D019087:Selectins / Q000276:immunology; D012822:Silicon Dioxide / Q000737:chemistry; D017945:Theilovirus / Q000276:immunology* / Q000472:pathogenicity
D000941:Antigens; D018941:Antigens, CD19; C000604812:CD19 antigen, mouse; D036022:Capsid Proteins; C476399:Cd44 protein, mouse; D024141:Collagen Type IV; D000939:Epitopes; D018960:Hyaluronan Receptors; D019087:Selectins; C442556:VP2 protein, Theilovirus; C016039:Percoll; D012822:Silicon Dioxide; D010047:Ovalbumin; D017364:Collagenases; D011205:Povidone
CD8 T cell; CNS viral infection; Lymphocyte isolation; Neuroinflammation; Optimized recovery; Theiler's murine encephalomyelitis virus