Given that nociceptor neuron secrete various neuropeptide and that NK cell expressed the receptor for these neuropeptide, we decided to devise a co-culture method to study the interplay between the NK cell and the nociceptor neurons. Such a reductionist method could be useful to study how nociceptor neuron control the anti-tumor function of NK cell, and it could also be interesting to study how NK cell controlled the elimination of injured neurons. Demonstrating the procedure will be Ali Ahmadi, a former master's student from my lab.
To begin the natural killer or NK cell isolation and culture, collect the mouse spleen in a 1.5 milliliter microcentrifuge tube containing 500 microliters of sterile PBS, then homogenize the spleen using a pestle. Next, dilute the cells with one milliliter of sterile PBS and filter the mixture through a 50 micron cell strainer into a 50 milliliter conical tube. Top up the filtered homogenate volume to 10 milliliters using sterile PBS.
Count the cells in 10 microliters of the diluted homogenate using a hemocytometer. Centrifuge the remaining cells for five minutes at 500 G and remove the supernatant. Then resuspend the cells at 10 to the eighth cells per milliliter in supplemented RPMI 1640 medium.
Magnetically purify the spleen NK cells using a mouse NK cell isolation kit and confirm NK cell purity using flow cytometry. Count the cells using a hemocytometer, followed by centrifugation at five minutes at 500 G, then remove the supernatant. For stimulating the pelleted NK cells, resuspend these cells in IL-2 and IL-15 supplemented RPMI 1640 culture medium.
Culture two times 10 to the sixth NK cells per milliliter in a 96-well U-bottom plate for 48 hours. 24 hours before the NK cell stimulation, coat the 96-well plate with 100 microliters per well of laminine and incubate it for 45 minutes at 37 degrees Celsius. After incubation, remove the solution and allow the wells to air dry in the biosafety cabinet.
Next, secure the euthanized mouse on the board in a prone position. Lift the skin and incise the skin along the dorsal column. Cut off the lumbosacral joint and separate the sacrum from the lumbar spine with scissors.
Then separate the spine by cutting the muscles and ribs on both sides in the cranial direction until the base of the skull is reached. Use scissors to cut off the spine at the atlantooccipital joint. Next, remove the muscle and fatty tissue from the spine.
Pin and open the vertebral column to remove the spinal cord before gaining access to the dorsal root ganglion or DRG in the intervertebral foramina connected to the spinal cord through the dorsal root. Next, harvest the DRGs into a 15 milliliter conical tube filled with 10 milliliters of ice cold supplemented DMEM. Then centrifuge the preparation for five minutes at 200 G at room temperature and remove the supernatant.
Add a 250 microliters of PBS containing one milligram per milliliter collagenase IV and 2.4 units per milliliter dispase II.Incubate this DRG with collagenase-dispase solution for 80 minutes with mild agitation. To inactivate the enzymes, add five milliliters of supplemented DMEM medium and centrifuge the solution at 200 G.After five minutes, gently remove the supernatant using a pipette and leave around 100 microliters of the supernatant to avoid unwanted cell loss. Next, add one milliliter supplemented DMEM medium to the cells and use a pipette gun and three glass Pasteur pipettes of decreasing sizes to gently triturate the DRG into a single cell preparation.
While working in a biosafety cabinet, dilute bovine serum albumin or BSA in sterile PBS to a final concentration of 15%and store one milliliter aliquots at minus 20 degrees Celsius. To create a BSA gradient in a 15 milliliter conical tube, add two milliliters of sterile PBS and then slowly dispense one milliliter of 15%BSA solution at the bottom of the tube. Avoid disrupting the gradient by gently removing the pipette.
Using a P200 pipette, add the triturated ganglia suspension containing the neurons onto the side of the tube into the gradient. Then set the acceleration and deceleration at the minimum to centrifuge the neuron containing the BSA gradient for 12 minutes at 200 G.Following the centrifugation, remove all the supernatant using a pipette and resuspend the cells in 500 microliters of Neurobasal. Plate 10, 000 neurons per well in a laminine-coated flat bottom 96-well plate.
To allow the attachment of the neurons, incubate the 96-well plate overnight at 37 degrees Celsius. To start co-culturing, slowly remove the Neurobasal from the neuron culture. After 48 hours of stimulation with IL-2 and IL-15, resuspend the NK cells and add 10 to the fifth NK cells per well to the neuron culture.
Co-culture the cells in supplemented Neurobasal MX at 37 degrees Celsius. After 48 hours, collect and wash the cells thrice with PBS and centrifuge them for five minutes at 500 G at four degrees Celsius. Discard the supernatant before staining the cells with one to 1, 000 diluted viability dye eFluor 780 for 15 minutes at four degrees Celsius.
Wash and centrifuge the cells as demonstrated, followed by treating with CD 1632 for 15 minutes at four degrees Celsius to block non-specific sites on the cells, followed by collecting and washing the cells with PBS thrice. After the next cycle of washing and centrifugation, stain the cells with BV421, anti-NK-1.1, fluorescein isothiocyanate anti-NKp46, and phycoerythrin anti-GM-CSF for 15 minutes. Once again, collect and wash the cells thrice with PBS, centrifuge them and discard the supernatant, then resuspend the cells in 500 microliters of FACS buffer and immunophenotype the NK cells using flow cytometry.
When cultured alone, NK cells expressed basal levels of granulocyte macrophage colony stimulating factor or GM-CSF and NKp46. When co-cultured with DRG neurons harvested from nociceptor intact mice, capsaicin stimulation decreased NK cells expression of GM-CSF. The capsaicin had no impact on NK cell activations when co-cultured with DRG neurons harvested from nociceptor ablated mice.
The level of NKp46 was unaffected. The key steps in this protocol is the DRG dissociation and isolation. The idea is to keep maximal neuronal viability by avoiding making bubble and using the right size pipette.
After the protocol, investigator can use RNA sequencing or qPCR to measure transcript change in FACS purified cells, or they can use ELISA to measure the secretion of cytokine or neuropeptides. Such interplay reveal that NK cell function is likely tuned by nociceptor neuron. And this crosstalk may be important in biological processes spanning from nerve injury, bacterial and viral infection, all the way to malignancies.
