Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.
Emerging evidence has implicated the contribution of dorsal root ganglion macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping the response of dorsal root ganglion macrophages is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol.
This protocol is far less time-consuming compared to standard enzymatic protocols, and we've routinely used it for our fluorescence-activated cell sorting analysis. Before starting the experiment, prepare the working solution of the density gradient medium by mixing nine volumes of the medium with one volume of calcium and magnesium-free 10X HBSS and keep it on ice. Confirm that the mouse is fully anesthetized by the lack of response to the hind paw pinch.
Begin by placing an anesthetized mouse inside a chemical fume hood in the supine position with four paws secured with tape. Use forceps to lift the skin below the ribcage. And then with surgical scissors, make a small incision to expose the liver and diaphragm.
Using the same scissors, cut the diaphragm and the ribcage. And then open the pleural cavity to expose the beating heart. Next with iris scissors, quickly cut the right atrial appendage.
Once the bleeding is noted, insert a 30-gauge needle into the posterior end of the left ventricle and slowly inject 10 milliliters of pre-chilled 1X PBS to perfuse the animal. To perform dorsal laminectomy, place the mouse in the prone position. Use a size 11 scalpel to make two longitudinal lateral deep incisions starting from the thoracic region down to the sacral region.
Then remove the skin exposing the dorsal muscle layer. Then with Friedman-Pearson rongeur, peel off the connective tissues and muscles until the lumbosacral spine processes and bilateral transverse processes are exposed. First, use a Friedman-Pearson rongeur to carefully open the dorsal spinal column, then switch between a Friedman-Pearson rongeur and Noyes spring scissors to remove the vertebral bones, exposing the spinal cord with intact spinal nerves attached.
Carefully dissect ipsilateral and contralateral lumbar DRG. Place it into one milliliter of ice cold calcium and magnesium-free 1X HBSS in a Dounce tissue homogenizer. Homogenize the DRG tissue in the Dounce homogenizer with a loose pestle for 20 to 25 times.
Place a sterile 70-micrometer nylon cell strainer in a sterile 50-milliliter conical tube. Use 800 microliters of ice code 1X HBSS to wet the cell strainer. Use a pipette to collect the homogenized tissue suspension from the homogenizer onto the wet cell strainer, and collect it into the 50-milliliter conical tube.
Rinse the homogenizer twice with 800 microliters of ice cold 1X HBSS, collecting the liquid into the same 50-milliliter conical tube to increase the yield. Add 1.5 milliliters of equilibrated ice cold isotonic density gradient medium into a sterile five-milliliter polystyrene flask tube. Transfer the cell homogenate from the 50-milliliter conical tube into this flask tube.
And pipette up and down to mix well. Add an additional 500 microliters of 1X HBSS to seal the top. Centrifuge the cells at 800 times G for 20 minutes at four degrees Celsius.
Carefully aspirate the supernatant containing myelin in the medium without disturbing the cell pellet at the bottom of the tube. Add 100 microliters of PBS containing 5%fetal bovine serum to the pellet, and re-suspend the DRG cells. Then add alpha mouse CX3CR1 APC antibody to the cells and incubate in the dark at four degrees Celsius for one hour.
Wash the cells once with five milliliters of PBS. Centrifuge the cells at 360 times G for eight minutes at four degrees Celsius. Aspirate the supernatant, and then re-suspend the cell pellet in 300 microliters of PBS for facts analysis.
After intraperitoneal injections of FK-binding protein dimerizer AP into MaFIA mice to deplete macrophages, there was a significant loss of GFP positive cells in the DRGs of the AP-treated mice compared to controlled vehicle treated mice. Facts analysis revealed a successful depletion of GFP high population in AP-treated mice and demonstrated the high quality of isolated cells. 4%of total isolated cells from the DRG of vehicle treated animal were GFP high macrophages.
In contrast only 0.4%of total DRG cells were GFP high macrophages in AP-treated mouse. Mechanically isolated DRG cells from wild type mice were stained with alpha mouse CX3CR1-APC antibody. 6%of the DRG cells were CX3CR1 positive macrophages.
When cell viability was assessed with propidium iodide, it revealed that more than 80%of freshly isolated DRG cells were viable. If the unsatisfying cell yield is noted, insufficient or excessive homogenization of DRG tissue may be suspected. In addition, DRG dissection may require repeated practice for beginners.
Likely the application of our protocol can be expanded to study other non-neuronal cells such as satellite cells and T cells. Further studies may be necessary to confirm the effectiveness of cell isolation.
