This protocol allows us to enhance the number of neurons in adult mouse dorsal root ganglia cultures. This may help determine the contribution of neuronal cells to a given response. We have added an immunopanning step to a basic DRG culture protocol.
The main advantage is to select against non-neuronal cells. If you're new to primary DRG cultures, it's best to practice dissections to get as many DRGs as possible, and trim as much of the nerve as possible. To begin, aspirate the poly-D-lysine used for coating the culture plate, and wash the plate three times with tissue culture water.
Tilt the lid open and allow the surface to dry completely. Apply enough laminin to the plate to coat the bottom of each well, and place it in an incubator at 37 degrees Celsius. Wash the previously prepared panning dishes of CD45, PDGFR-beta, and O4 with D-PBS.
For the CD45 dish, add 5 milliliters of 0.2%BSA and 20 microliters of CD45 primary antibody. For the PDGFR-beta dish, add 15.2 microliters of PDGFR-beta to the 5 milliliters of 0.2%BSA-containing plate. For the O4 dish, add 2 milliliters of O4 hybridoma to 3 milliliters of 0.2%BSA, and an additional 100 microliters of 4%BSA, to ensure the final BSA concentration remains at 0.2%Next, perfuse the euthanized mouse with 30 milliliters of 0.9%ice-cold saline.
Observe the change in color of the liver to ensure perfusion. Pin the front and hind paws to a styrofoam stage, and use a clean razor blade to expose the spinal column from the back. Perform a laminectomy by making cuts about halfway deep on either side of the dorsal spinal column, removing the dorsal half of the column to expose the spinal cord.
Either gently cut the nerves and remove the spinal cord, or gently push the cord to the side, maintaining nerve integrity. Under a dissection microscope, use the spinal nerve roots to find and remove all the dorsal root ganglia, or DRGs, from either side of the spinal column. Trim the nerve debris from each DRG as it is removed, since more nerve debris in the culture may lead to poor survival.
Collect the DRGs in 15 milliliters of HBSS in a conical tube on ice, and allow them to settle to the bottom. Using a pipette, aspirate most of the HBSS from the DRGs, and leave approximately 100 microliters of liquid in the tube, to avoid losing the tissue. Wash three times with 1 milliliter of HBSS, and add 5 milliliters of previously thawed working STEMxyme solution to the tissue.
Cover the lid with a transparent film, and float the tube on its side in a 37 degrees Celsius water bath, for one hour. After incubation with the enzyme, add 1 milliliter of low ovomucoid inhibitor solution to the tube. Triturate the cells gently with a P1000 pipette 10 to 15 times, and allow the tissue chunks to settle.
Then transfer the top 2 to 3 milliliters of the solution containing dissociated cells to fresh low ovomucoid solution, and repeat until the tissue is fully dissociated, and no visible chunks remain. Centrifuge the tube for 10 minutes at 300 x g at room temperature. After removing the supernatant using a pipette, as demonstrated earlier, resuspend the cell pellet in 1 milliliter of panning buffer, and gently pipette to mix.
Pre-wet a 70-micrometer cell strainer with 1 milliliter of panning buffer over a 50 milliliter conical tube, and then filter the cell solution through the cell strainer. Wash the tube with 1 milliliter of panning buffer, and pass it through the strainer. For cell layering, add 2 milliliters of 15%BSA and gently coat the sides of the closed tube.
To remove the myelin debris, carefully layer the cell suspension one milliliter at a time by pipetting against the side of the tube, on top of the BSA cushion. Centrifuge the tube at 300 x g for 10 minutes at room temperature, with slow acceleration and deceleration. The middle myelin layer shows flakes of white material.
Using a P1000 pipette, remove the clear liquid on top, the myelin phase in between, and the BSA, one milliliter at a time, leaving approximately 100 microliters so as not to disturb the pellet. For antigen retrieval, add 5 milliliters of panning buffer and incubate the tube in a 37 degrees Celsius and 10%carbon dioxide incubator for 30 to 45 minutes. Next, wash the incubated CD45 dish three times with D-PBS, and pour off the final rinse.
Then pour the cell suspension into the CD45 dish. Incubate the dish for 20 minutes at room temperature, swirling gently at 10-minute intervals to allow the cells to gain equal access to the antibody. Gently shake the CD45 dish and prop it up at an angle.
Carefully pipette 1 milliliter of the cell suspension over the dish, to collect unbound cells. Then transfer it to the PDGFR-beta dish, previously washed three times with D-PBS, and incubate. Transfer the unbound cells from the PDGFR-beta dish to the O4 dish, as demonstrated earlier, and incubate the plate.
After the incubation, transfer the cell suspension to a 15-milliliter conical tube and centrifuge at 300 x g for 10 minutes. Resuspend the cells in the desired volume, and dilute to a ratio of 1:1 with trypan blue for cell viability. Using a hemocytometer, count the medium to large cells.
Remove the laminin and immediately plate the cells at the desired density, without allowing them to dry before incubating the plate. The fixed, whole, and immunopanned DRG cultures were stained with beta3-tubulin for neurons and DAPI for all nuclei. Whole DRG cultures were determined to have approximately 42.36%beta3-tubulin staining, and immunopanned DRG cultures were determined to have approximately 71.44%beta3-tubulin staining, which reveals a significant increase in neuronal enrichment with immunopanning.
Treating the tissue with care is very important. Handling DRGs gently, trimming excess nerve, and taking care not to agitate or lose tissue and culture are all crucial to be aware of.
