Isolating Sensory Neurons and Co-Culturing with Natural Killer Cells

研究方案

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

1. NK cell isolation, culture, and stimulation

1. Generate nociceptor neuron intact (littermate control; TRPV1wt::DTAfl/wt) and ablated (TRPV1cre::DTAfl/wt) mice by crossing lox-stop-lox-diphtheria toxin mice (DTAfl/fl) with TRPV1cre/wt mice.
2. Using CO2 inhalation, euthanize a naïve littermate control (TRPV1wt::DTAfl/wt) mouse.
3. Collect the spleen in a 1.5 mL microcentrifuge tube containing 500 µL of sterile phosphate-buffered saline (PBS).
4. Homogenize the spleen using a pestle.
5. Dilute the cells with 1 mL of sterile PBS.
6. Filter the mixture through a 50 µm cell strainer into a 15 mL conical tube.
7. Top up the volume (10 mL) with sterile PBS.
8. Collect 10 µL and count the cells using a hemocytometer.
9. Centrifuge the cells (500 × g, 5 min) and remove the supernatant.
10. Resuspend the cells at a concentration of 10⁸ cells/mL in a supplemented RPMI 1640 medium.
11. Follow the manufacturer's instructions to magnetically purify spleen Natural Killer (NK) cells using a mouse NK cell isolation kit. Confirm NK cell purity using flow cytometry.
12. Collect 10 µL and count the cells using a hemocytometer.
13. Centrifuge the cells (500 × g, 5 min) and remove the supernatant.
14. Resuspend the NK cells in a supplemented RPMI 1640 culture medium (containing IL-2 and IL-15).
15. Culture 2 × 10⁶ NK cells/mL in a 96-well U-bottom plate for 48 h.

2. DRG neuron isolation and culture

1. At 24 h prior to the end of the NK cell stimulation (step 1.15), coat a 96-well plate with 100 µL/well of laminin (1 ng/mL) and incubate for 45 min (37 °C).
2. After incubation, remove the solution and allow the wells to air-dry in a biosafety cabinet.
3. Using CO2 inhalation, euthanize nociceptor neuron intact (littermate control; TRPV1wt::DTAfl/wt) or ablated (TRPV1cre::DTAfl/wt) mice.
   NOTE: CO2 inhalation is preferred to cervical dislocation as it avoids disrupting the spinal nerves connected to the DRG (Dorsal root ganglion).
4. Secure the mouse on the board in a prone position, lift the skin, and incise the skin along the dorsal column.
5. Cut off the lumbosacral joint and separate the sacrum from the lumbar spine with scissors.
6. Separate the spine by cutting the muscles and ribs on both sides of the spine in the cranial direction until the base of the skull is reached.
7. Using scissors, cut off the spine at the atlanto-occipital joint.
8. Remove muscle and fatty tissue from the spine.
9. Pin and open the vertebral column to remove the spinal cord and gain access to the DRG. Look for the DRG in the intervertebral foramina connected to the spinal cord through the dorsal root but separated from the meninges.
10. Harvest the DRGs into a 15 mL conical tube filled with 10 mL of ice-cold supplemented DMEM.
11. Centrifuge the preparation (200 × g, 5 min, room temperature) and remove the supernatant.
12. Add 250 µL of PBS containing Collagenase IV (1 mg/mL) and Dispase II (2.4 U/mL).
13. Incubate the DRG with Collagenase IV/Dispase II (C/D) solution (80 min, 37 °C, mild agitation). When pooling ganglia from several mice, maintain a ratio of 125 µL of C/D solution per ganglion.
14. To inactivate the enzymes, add 5 mL of supplemented DMEM(Dulbecco's Modified Eagle Medium) medium.
15. Centrifuge the solution (200 × g, 5 min) and gently remove the supernatant using a pipette.
16. To avoid unwanted cell loss, leave ~100 µL of the supernatant.
17. Add 1 mL of supplemented DMEM medium.
18. Using a pipette gun and three glass Pasteur pipettes of decreasing sizes, gently triturate (~10 up/down per Pasteur pipette size) the DRG into a single-cell preparation.
19. In a biosafety cabinet, dilute bovine serum albumin (BSA) in sterile PBS (Phosphate buffered saline) to a final concentration of 15%. Store 1 mL aliquots at −20 °C.
20. Create a BSA gradient in a 15 mL conical tube by adding 2 mL of sterile PBS and slowly dispensing 1 mL of 15% BSA solution at the bottom of the tube. Avoid disrupting the gradient by gently removing the pipette.
21. Using a P200 pipette, slowly pipette the triturated ganglia suspension (which contains the neurons) onto the side of the tube into the gradient.
22. Centrifuge the neuron-containing BSA gradient (200 × g, 12 min, room temperature). Set the acceleration and deceleration of the centrifuge to the minimum speed.
    NOTE: After centrifugation, the neurons will be at the bottom of the tube, while the cell debris will be trapped in the BSA gradient.
23. Gently remove all of the supernatant using a pipette.
24. Resuspend the cells in 500 µL of Neurobasal.
25. Plate 10⁴ neurons per well (200 µL of neurobasal/well) in a laminin-coated, flat-bottom 96-well plate.
    NOTE: On average, an investigator will be able to fill ~8 wells per mouse.
26. Culture the neurons (37 °C, overnight) to allow attachment.

3. Co-culture

1. Slowly remove the neurobasal from the neuron culture.
2. After 48 h stimulation with IL-2 and IL-15 (see steps 1.14-1.15), resuspend the NK cells and add 10⁵ NK cells/well to the neuron culture (96-well flat bottom plate).
   NOTE: On average, the investigator will be able to fill ~6 wells per mouse.
3. Co-culture the cells in supplemented neurobasal MX (37 °C, 48 h).

材料

Name	Company	Catalog Number	Comments
B-27	Jackson Laboratory	Cat no: 009669
Bovine Serum Albumin (BSA) culture grade	World Precision Instruments	Cat no: 504167
BV421 anti-mouse NK-1.1	Fisher Scientific	Cat no: 12430112
Cell strainer (50 μm)	Fisher Scientific	Cat no: A3160702
Collagenase IV	Fisher Scientific	Cat no: 15140148
Dispase II	Fisher Scientific	Cat no: 13-678-20B
Dulbecco's Modified Eagle Medium (DMEM)	Fisher Scientific	Cat no: 07-200-95
EasySep Mouse NK Cell Isolation Kit	Sigma	Cat no: CLS2595
Ethylenediaminetetraacetic acid (EDTA)	Sigma	Cat no: C0130
Fetal bovine serum (FBS)	Sigma	Cat no: 806552
Glass Pasteur pipette	Sigma	Cat no: 470236-274
Glial cell line-derived neurotrophic factor (GDNF)	VWR	Cat no: 02-0131
Laminin	Cedarlane	Cat no: 03-50/31
L-Glutamine	Gibco	Cat no: A14867-01
Mouse recombinant IL-15	Gibco	Cat no: 22400-089
Mouse recombinant IL-2	Gibco	Cat no: 21103-049
Nerve Growth Factor (NGF)	Life Technologies	Cat no: 13257-019
Neurobasal media	PeproTech	Cat no: 450-51-10
Penicillin and Streptomycin	PeproTech	Cat no: 210-15
Pestles	Stem Cell Technology	Cat no: 19855
Phosphate Buffered Saline (PBS)	Biolegend	Cat no: 108732	Clone PK136
RPMI 1640 media	Biolegend	Cat no: 137606	Clone 29A1.4
Tweezers and dissection tools.	Biolegend	Cat no: 65-0865-14
U-Shaped-bottom 96-well plate	Biolegend	Cat no: 101319
Viability Dye eFlour-780	Becton Dickinson
CO2 Incubator
Dissection microscope