Isolating Invariant Natural Killer T Cells from a Mouse Model

Abstract

Source: Meng, M., et al. Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice. J. Vis. Exp. (2020)

This video illustrates a method for isolating invariant natural killer T cells, also known as iNKT cells. The procedure begins with injecting a synthetic glycolipid into a mouse to stimulate the activation and proliferation of iNKT cells in the spleen. Subsequently, single-cell suspensions are prepared from the spleen and incubated with iNKT-cell-recognizing complexes labeled with fluorophores, followed by magnetic bead-based separation, to isolate the iNKT cells.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Obtaining invariant natural killer T cells (iNKT Cells) with Adoptive Cellular Therapy

1. Directional induction of iNKT cells
   1. Inject normal mice intraperitoneally with α-Galactosylceramide (α-GalCer) (0.1 mg/kg of body weight).
2. Isolation of iNKT cells
   1. Three days after modeling, inject mice intraperitoneally with 1% sodium pentobarbital (50 mg/kg of body weight) for anesthetization. Adequately anesthetized mice do not show a hind paw withdrawal response to a toe pinch.
   2. Isolate the spleen of a DBA/1 mouse after injecting it intraperitoneally with α-GalCer. Prepare a single-cell suspension by cutting and grinding the spleen in a 200-mesh sieve.
   3. Wash the cell suspension with phosphate-buffered saline (PBS), centrifuge at 200 x g for 5 min, and discard the supernatant. Repeat.
   4. Resuspend the cells with 1 mL of whole blood and tissue dilution solution. Add 3 mL of mouse lymphocyte separation medium, then centrifuge the cells for 20 min at 300 x g at room temperature.
   5. Collect the layer of milky white lymphocytes (i.e., the second layer from the top), wash it 2x with PBS, and count with an automated cell counter.
3. Magnetic activated cell sorting (MACS) positive selection strategy for purification of iNKT cells
   NOTE: For the pretreatment of CD1d tetramers, 1 mg/mL of α-Galcer was diluted to 200 μg/mL with 0.5% of Tween-20 and 0.9% of NaCl, and 5 µL of the resulting solution was added to 100 µL of the CD1d tetramer solution. The mixture was incubated for 12 h at room temperature and placed at 4 °C for use. T cell receptor (TCR) β was diluted 80x with deionized water. All other antibodies were used as a stock solution.
   1. Resuspend 10⁷ cells with 100 µL of 4 °C PBS, add 10 µL of α-GalCer-loaded CD1d Tetramer-phycoerythrin (CD1d Tetramer-PE), and incubate them at 4 °C for 15 min in the dark.
   2. Wash the cells 2x with PBS and resuspend them in 80 µL of PBS.
   3. Add 20 µL of anti-PE-MicroBeads and incubate them at 4 °C for 20 min in the dark.
   4. Wash them 2x with PBS and resuspend the cells with 500 µL of PBS.
   5. Place the sorting column in the magnetic field of the MACS sorter and rinse with 500 µL of PBS.
   6. Add the cell suspension from step 1.3.4 to the sorting column, collect the flowthrough, and rinse 3x with PBS buffer.
   7. Remove the magnetic field and collect the cells from the sorting column. At this point, add 1 mL of PBS buffer to the sorting column and quickly push the plunger at a constant pressure to drive the labeled cells to the collection tube and obtain purified iNKT cells. Count with an automated cell counter.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-PE MicroBeads	Miltenyi	130-048-801	Germany
Columns	Miltenyi	MS	Germany
Cryogenic Centrifuge	Beckman	Allegra® X-15R	America
Mouse CD1d Tetramer-PE	MBL	TS-MCD-1	Japan
Mouse percoll	Solarbio	P8620	China
Optical Microscope	Olympus	Olympus-II	Japan