Establishing a Primary Dorsal Root Ganglia Cell Culture from a Rat Spinal Column
Transcrição
Collect the lumbar dorsal root ganglia from the trunk of a two to three-week-old rat. Begin by making two cuts along the sides of the spinal column and one lateral cut to mark the rostral extent of the lumbar spine. Then, use bone-cutting forceps to remove the dorsal muscles of the spine. Next, remove the dorsal portion of the vertebrae and expose the spinal cord, then use dissection scissors and forceps to remove the spinal cord.
Identify the lumbar DRG by counting vertebrae from the last rib. This diagram shows the vertebrae positions. Use micro-scissors to collect the 12 bilateral lumbar DRG from L1 to L6. Remove any attached neuronal fibers to improve the purity of the culture. Transfer each lumbar DRG to a 35-millimeter culture dish containing 2 milliliters of ice-cold serum-free medium.
Transfer the DRG-containing 35-millimeter dish into a laminar hood, and wash the DRG with serum-free medium three times by pipette. Use sterile tweezers to move the DRGs to a new 35-millimeter culture dish containing 2 milliliters of sterile collagenase type 1A. Place the tissue in collagenase solution in the 37 degrees Celcius tissue culture incubator for 30 minutes to begin dissociation of the cells.
Following the incubation, remove the collagenase solution, and wash the DRG three times in 2 milliliters of Hank's balanced salt solution. Next, add 2 milliliters of pre-warmed 0.05% trypsin-EDTA to the dish, and digest the DRG in the incubator for 30 minutes as before. After digestion, use a glass pipette to transfer the 2 milliliters of DRG-containing solution to a 15-milliliter centrifuge tube.
The DRG might stick to the glass pipette so this step should be performed with care. DRG loss can be avoided by keeping the DRG-containing solution in the tapered end of the glass pipette and transferring the solution into the centrifuge tube slowly but without pause.
Next, centrifuge the solution at 290 times g for 5 minutes at 4 degrees Celsius. After centrifuging, remove the supernatant, and add another 2 milliliters of serum-free medium to resuspend the DRG. Repeat the wash twice using 2 milliliters of pre-warmed culture medium to resuspend the tissue after the final centrifugation.
Use the previously prepared sterile flame-polished pipette to manually triturate the DRG approximately 60 times. Next, remove a poly-L-lysine-coated 24-well plate containing 1 milliliter of culture medium per well from the CO2 incubator. Aspirate the incubated culture medium from the dish.
Seed the DRG cells from one rat into four of the wells. This gives a seeding density of approximately 500,000 cells per well. The following day, replace the culture medium with medium supplemented with 10 micromolar AraC, and 100 nanograms per milliliter NGF.
