This article describes a method for isolating sensory neurons from dorsal root ganglia (DRG) for co-culture studies with natural killer (NK) cells. The protocol involves enzymatic dissociation, density gradient centrifugation, and subsequent culture techniques.
Take a dorsal root ganglion or DRG, a cluster of sensory neurons, in a cold, serum-containing medium to protect the neurons during subsequent processing.
Centrifuge, then remove the supernatant.
Add a phosphate buffer containing digestive enzymes and incubate with mild agitation.
The enzymes dissociate the extracellular matrix, or ECM, and DRG tissue, loosening the cells.
Add a serum-containing medium to inactivate the enzymes.
Centrifuge, then discard the supernatant.
Resuspend the DRG and mechanically dissociate the tissue using pipettes of progressively smaller sizes to obtain a cell suspension.
Layer the cells onto a density gradient of bovine serum albumin.
Centrifuge. The neurons settle to the bottom, forming a pellet.
Discard the layers containing tissue debris.
Resuspend the neurons in a culture medium.
Add the neurons to an ECM-coated culture dish and incubate.
Remove the medium and add natural killer or NK cells.
Add a medium to co-culture and study the interaction between these cells.
Harvest the DRGs into a 15-milliliter conical tube filled with 10 milliliters of ice-cold, supplemented DMEM. Then, centrifuge the preparation for 5 minutes at 200 g at room temperature, and remove the supernatant. Add 250 microliters of PBS containing 1 milligram per milliliter of collagenase IV and 2.4 units per milliliter Dispase too. Incubate this DRG with collagenase dispase solution for 80 minutes with mild agitation.
To inactivate the enzymes, add 5 milliliters of supplemented DMEM medium and centrifuge the solution at 200 g. After 5 minutes, gently remove the supernatant using a pipette and leave around 100 microliters of the supernatant to avoid unwanted cell loss. Next, add 1 milliliter supplemented DMEM into 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.
Using a P200 pipette, add the triturated ganglia suspension containing the neurons onto the side of the tube into the BSA 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 laminin-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 and add 105 NK cells per well to the neuron culture.
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