Mice were euthanized by CO2 induced asphyxiation and perfused with 1X Dulbecco's PBS (CellGro) through the left ventricle with drainage mediated by laceration of the inferior vena cava. From individual mice, single cell suspensions of the CNS, brain and spinal cord, were generated by passing the tissue through a 100μm strainer. Mononuclear cells were isolated by Percoll density centrifugation (Sigma-Aldrich). CD4+ T cells were isolated from pooled CNS mononuclear cells using MACS with L3T4 CD4 positive selecting beads (Miltenyi Biotec). Enriched T cells were assessed for antigen specificity and effective 2D affinities using the 2D micropipette adhesion frequency assay using pMHC coated RBC sensors (25 (link), 27 (link), 35 (link)-37 (link)).
Protocol detail
Isolation and Characterization of CNS-infiltrating CD4+ T cells
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Antigen specificity
Asphyxiation
Assay
Brain
Cd4 t cells
Cells
Centrifugation
Drainage
Inferior vena cava
Laceration
Left ventricle
Mice
Percoll
Spinal cord
T cells
Tissue
Corresponding Organization : University of Utah
Other organizations : Emory University, University of California, San Francisco
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Variable analysis
independent variables
- Method of euthanasia (CO2 induced asphyxiation)
- Perfusion method (left ventricle with drainage through inferior vena cava)
- Tissue processing (passing through 100μm strainer)
- Cell isolation method (Percoll density centrifugation)
- Cell enrichment method (MACS with CD4 positive selecting beads)
- Antigen specificity of enriched T cells
- Effective 2D affinities of enriched T cells
- Dulbecco's PBS (CellGro) for perfusion
- Percoll density centrifugation
- MACS with CD4 positive selecting beads
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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