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

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

Abstract

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.

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

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Antigens / immunology*
  • Antigens, CD19 / immunology
  • B-Lymphocytes / immunology
  • Brain / immunology*
  • Brain / metabolism
  • CD8-Positive T-Lymphocytes / immunology*
  • Capsid Proteins / immunology
  • Cardiovirus Infections / immunology*
  • Cardiovirus Infections / virology
  • Cell Separation / methods*
  • Cell Survival
  • Centrifugation, Density Gradient
  • Collagen Type IV / metabolism
  • Collagenases / metabolism
  • Disease Models, Animal
  • Epitopes
  • Female
  • Flow Cytometry
  • Hyaluronan Receptors / immunology
  • Lymphocyte Activation*
  • Male
  • Mice, Inbred C57BL
  • Ovalbumin / immunology*
  • Povidone / chemistry
  • Selectins / immunology
  • Silicon Dioxide / chemistry
  • Theilovirus / immunology*
  • Theilovirus / pathogenicity

Substances

  • Antigens
  • Antigens, CD19
  • CD19 antigen, mouse
  • Capsid Proteins
  • Cd44 protein, mouse
  • Collagen Type IV
  • Epitopes
  • Hyaluronan Receptors
  • Selectins
  • VP2 protein, Theilovirus
  • Percoll
  • Silicon Dioxide
  • Ovalbumin
  • Collagenases
  • Povidone