Encyclopedia of Experiments: Immunology
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Transcript
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.
