This article details a protocol for isolating and enriching neuronal cultures from dorsal root ganglia (DRG) cells. The method involves immunopanning techniques to selectively remove non-neuronal cells and obtain a pure neuronal population.
Begin by layering the dorsal root ganglia-derived cells onto bovine serum albumin or BSA.
The cell suspension contains neurons, immune cells, fibroblasts, Schwann cells, and fatty myelin debris.
Centrifuge. The denser DRG cells pass through, forming a pellet.
Remove the myelin layer along with the serum.
Resuspend the cells in an immunopanning buffer, which improves the accessibility of the antigen for antibodies.
Take Petri dishes coated with secondary antibodies conjugated with cell-specific primary antibodies.
Add the cell suspension to the immune cell-specific antibody-coated Petri dish.
The immune cells attach to the antibodies while the other cells remain unbound.
Transfer the remaining cells to the next Petri dish to remove the fibroblasts.
Repeat the procedure to remove the Schwann cells.
Plate the unbound cells onto a matrix-coated multiwell plate containing a neurobasal medium and incubate to obtain an enriched neuronal culture.
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 1 milliliter at a time by pipetting against the side of the tube, on top of the BSA cushion.
Centrifuge the tube at 300 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, 1 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 DPBS, 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 DPBS, 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. Remove the laminin, and immediately, plate the cells at the desired density without allowing them to dry before incubating the plate.
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