This article describes a method for isolating invariant natural killer T (iNKT) cells from mouse spleen tissue. The process involves the use of a synthetic glycolipid and specific labeling techniques to purify the iNKT cells for further study.
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.
Inject normal DBA/1 mouse intraperitoneally with alpha-GalCer, at the dosage of 0.1 milligrams per kilogram of body weight. Three days after modeling, after anesthetization, isolate the spleen of the mouse. Prepare a single-cell suspension by cutting and grinding the spleen in a 200-mesh sieve. Wash the cell suspension with PBS. Centrifuge at 200 times g for 5 minutes, and discard the supernatant.
Resuspend the cells with 1 milliliter of whole blood and tissue dilution solution. Add 3 milliliters of mouse lymphocyte separation medium, and then centrifuge the cells for 20 minutes, at 300 times g, at room temperature.
To purify the iNKT cells, resuspend 10 to the 7 cells with 100 microliters of 4 degrees Celsius PBS. Add 10 microliters of alpha-GalCer-loaded CD1d Tetramer-PE and incubate them at 4 degrees Celsius for 15 minutes in the dark. Wash the cells twice with PBS, and resuspend them in 80 microliters of PBS. Add 20 microliters of anti-PE microbeads and incubate them at 4 degrees Celsius for 20 minutes in the dark. Wash them twice with PBS and resuspend the cells with 500 microliters of PBS.
Place the sorting column in the magnetic field of the MACS sorter, and rinse with 500 microliters of PBS. Add the prepared cell suspension to the sorting column, collect the flow-through, and rinse three times with PBS buffer. Remove the magnetic field and add 1 milliliter of PBS buffer to the sorting column. Quickly push the plunger at a constant pressure to drive the labeled cells into the collection tube, and obtain the purified iNKT cells. Count with an automated cell counter.
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