Encyclopedia of Experiments: Immunology
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Transcript
Begin by intraperitoneally injecting a synthetic glycolipid into a mouse.
Antigen-presenting cells, or APCs, internalize the glycolipid and present it via the non-classical MHC molecule CD1d.
The APCs migrate to the spleen and interact with the invariant natural killer T cells or iNKT cells, inducing iNKT cell activation and proliferation.
Harvest the spleen. Mechanically dissociate it to generate a single-cell suspension.
Transfer the cells to a tube. Add a density gradient medium and centrifuge to separate the lymphocytes.
Collect the lymphocytes. Add glycolipid antigen-loaded fluorophore-labeled CD1d tetramers that interact with the iNKT cells.
Remove unbound tetramers. Overlay with magnetic microbeads that bind to the tetramer-linked fluorophores.
Remove unbound beads and load the cell suspension onto a magnetic column.
Under the magnetic field, the bead-bound iNKT cells are retained in the column while the remaining cells flow through.
Remove the column from the magnetic field. Add an elution buffer to elute the iNKT cells.
