This method can help to answer key questions in the sensory research field, such as peripheral nociception. The main advantage of this technical is that investigating the cellular mechanism of sensory neurons with the model that most simulates to physiological condition. 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 two 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 two milliliters of sterile collagenase type 1A. Place the tissue in collagenase solution in the 37 degree Celsius 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 two milliliters of Hank's balanced salt solution.
Next, add two milliliters of prewarmed 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 two 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.
The actual loss can be avoid by keeping the DRG-containing solution in the taper 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 five minutes at four degrees Celsius. After centrifuging, remove the supernatant and add another two milliliters of serum-free medium to resuspend the DRG.
Repeat the wash twice, using two milliliters of prewarmed 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 one 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. On day three after cell plating, change the medium to 0.5 milliliters of prewarmed serum-free medium, and incubate for one hour. During the incubation, add 50 millimolar siRNA in one microliter of RNase-free water to 12.5 microliters of serum-free medium per transfection.
Then pipette 2.5 microliters of transfection reagent into 10 microliters of serum-free medium for each transfection. Mix both solutions by pipette, and incubate this mixed transfection solution for 10 minutes at room temperature. After 10 minutes, add the transfection solution into the wells of the DRG-containing 24 well plate and mix by shaking gently.
Following a six hour incubation, add 0.5 milliliters of culture medium with 20%FBS, 10 micromolar AraC, and 100 nanograms per milliliter NGF into each well of cells. Finally, incubate the DRG in a 37 degree Celsius CO2 incubator for another 66 hours. On day six after plating, and 72 hours after siRNA transfection, change the culture medium to 200 microliters of serum-free medium.
Following a 30 minute incubation, add one microliter of the stimulation chemical and gently mix by pipetting. The NPFFR2 agonist, dNPA, and corresponding vehicle are used here. After incubating for the required period, collect the culture medium from the culture dish, and centrifuge at 5000 times G for five minutes at four degrees Celsius to remove any suspended impurities.
Collect the supernatant from the centrifugation and dilute with phosphate-buffered saline as needed. Assay the levels of neurotransmitters with commercially available enzyme immunoassay kits. On day one, the cell body of a single neuron indicated by the arrow was attached on the bottom of a dish.
A glial cell indicated by an arrowhead is also present. The glial cells duplicated and extended processes to surround the cell body of the sensory neuron on day two, a process that was even more prominent on day three. In another culture, CGRP protein was stained to reveal the shape of neurons.
CGRP protein staining appears in the cytoplasm and axons of sensory neurons. The nuclear morphologies of neurons and glial cells are distinct when stained with DAPI. The neurons have a larger and more rounded nucleus than glial cells.
By comparison, the nuclei of glial are more oval in shape. Once mastered, this technique can be done in two and half hours if it is performed properly. After watching this video, you should have a good understanding of how to dissect and culture the dorsal root ganglion, and use it as a tool to investigate underlying physiological functions.
Don't forget that working with primary cells can be much more difficult than working with regular cell lines, and being gentle is always the best way when performing these procedure.
