Encyclopedia of Experiments: Immunology
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Transcript
Macerate mouse spleen and lymph nodes on a cell strainer. Flush the strainer with a suitable buffer to collect splenocytes and lymphocytes.
Incubate the cells with a biotinylated antibody cocktail and magnetic anti-biotin microbeads that bind to the non-CD8+ cells.
Pass the cell suspension through a magnetic column. Owing to the magnetic force, the bead-bound non-CD8+ cells bind to the column wall while the CD8+ T cells elute out.
Incubate the eluted cells with artificial antigen-presenting cells, aAPCs. These are magnetic nanoparticles functionalized with antigen-loaded major histocompatibility complex-immunoglobulin fusion protein, MHC-Ig, and anti-CD28 antibody.
The antigen-loaded MHC-Igs bind to distinct receptors on the antigen-specific CD8+ T cells. The anti-CD28 antibodies interact with the T cell CD28 receptors, initiating T cell activation.
Place the tube containing magnetic nanoparticles and cell mixture on a magnetic stand. The aAPC-bound CD8+ T cells get attracted to the magnetic field, while non-specific CD8+ T cells remain suspended. Remove the supernatant.
Remove the tube from the magnetic stand. Resuspend the aAPC-bound cells in T cell growth factor-containing media. Seed them in a multi-well plate.
The growth factors induce antigen-specific CD8+ T cell expansion.
