Developing a Neuron and Macrophage Coculture In Vitro

1. Culture Preparation of Dissociated Adult dorsal root ganglion Neuron or DRG Neuron

1. Before setting up the culture, pre-coat a 6-well plate with poly-D-lysine and laminin. Incubate a 6-well plate with 0.01% poly-D-lysine at 37°C for 2 h or at 4°C overnight. Then, wash the plate twice with distilled water.
2. Incubate the plate with laminin solution at a concentration of 3 µg/mL for 2 h at room temperature, and then wash the plate twice with distilled water. Dry the plate at room temperature for at least 1 h.
3. Euthanize a mouse in a transparent CO₂ chamber connected to a compressed CO₂ gas cylinder. Incise the skin overlying the vertebral column with a surgical blade and dissect the paravertebral muscles bilaterally to expose the vertebral bones. Remove the vertebral bones meticulously using a narrow-tipped surgical rongeur until the DRGs are fully exposed.
   NOTE: Adult DRG neurons are obtained from adult C57BL6 male mice aged between 8 weeks and 12 weeks old.
4. Remove the DRGs bilaterally from the S1 all the way up to the C1 level using iridectomy scissors and fine-tipped forceps under a dissecting microscope. Try to cut out the roots entirely from DRGs in order to minimize the contamination of Schwann cells or fibroblasts.
5. Store the dissected DRGs in a 60 mm Petri dish containing 5 mL of cold Dulbecco's Modified Eagle Medium (DMEM) on ice until all the DRGs are the next step, even if there are still remaining DRGs are collected.
   NOTE: It is critical to dissect the DRGs as quickly as possible. The collection of all DRGs from one mouse should not take longer than 30 min. 30 min after the euthanasia, stop collecting the DRGs and proceed to Transfer the DRGs to a 1.5 mL Eppendorf tube using a blue pipette tip (1000 µL) with a cut-off end. Remove the DMEM after a quick spin for several seconds using a mini centrifuge. Add 1 mL of DMEM containing 125 U/mL type XI collagenase and incubate the tube for 90 min with a gentle rotation (35-40 rpm) using a twister shaker in a 37 °C incubator. Immobilize the tube on the shaker floor using the tape.
6. Discard collagenase-containing DMEM and add 1 mL of fresh DMEM. Wait until the floating DRGs completely sink down to the bottom, and then remove the DMEM with the tissue debris. Repeat this washing step at least six times.
   NOTE: Do not try to discard the supernatant completely. Be careful not to remove any DRGs with the supernatant DMEM.
7. Transfer the DRGs to a 15 mL conical tube using a blue pipette tip (1000 µL) with a cut-off end. Then pipette up and down gently at least 15 times using a blue tip (1000 µL) to make a homogeneous cell suspension. Avoid making bubbles and try not to touch the bottom of the conical tube with a pipette tip.
8. Centrifuge the tube at 239 x g for 3 min and carefully discard the supernatant with floating debris. Add 1 mL of Neurobasal medium supplemented with B27 (2.0% v/v) and resuspend the cell pellet by gently pipetting up and down 5 to 10 times.
9. Pass the cell suspension through a 70-µm cell strainer overlaid on top of a 50 mL conical tube. Wait for 2 min, and then pour Neurobasal/B-27 medium onto the strainer using a pipette controller.
10. Plate all collected DRG neurons ( approximately 2 x 10⁶ DRG neurons from one mouse) onto two wells of 6-well plate. DRG neurons from one adult mouse cover two wells in a 6-well plate. Then place the plate in a CO₂ incubator at 37°C until the macrophage co-cultures.

2. Co-culture of Primary Peritoneal Macrophages on A Cell Culture Insert

NOTE: Establish the co-cultures 4 h after the initial plating of the dissociated DRG neurons

1. Primary peritoneal macrophages are prepared from adult C57BL6 male mice aged between 8 weeks to 12 weeks old. Euthanize an animal in a CO2 chamber. Incise the abdominal skin delicately, expose the peritoneum, and avoid cutting the peritoneum to prevent leakage of lavage fluid.
2. Puncture the peritoneum using a syringe with a 22G needle and inject 10 mL of ice-cold phosphate-buffered saline (PBS) into the peritoneal cavity. Gently massage the peritoneum for 1-2 min. Then, pull out the needle and squeeze out the PBS through the needle puncture site, and collect the lavage fluid in a 50 mL conical tube.
   NOTE: Before use, put the syringe on ice to maintain the coldness of the PBS.
3. Centrifuge the lavage fluid at 239 x g for 10 min at 4 °C to pellet the cellular components. Resuspend the pellet with 3 mL of the red blood cell (RBC) lysis buffer for 3 min at room temperature. Then centrifuge the cell suspension again at 239 x g for 10 min at 4 °C. Resuspend the pellet with 3 mL of PBS and centrifuge the cell suspension again to remove any remaining RBC lysis buffer.
   NOTE: Make sure the RBC lysis buffer is completely removed. Otherwise remaining RBC lysis buffer may be toxic to the cultured macrophages.
4. Resuspend the pelleted cells in 1 mL of Neurobasal/B-27 medium. Plate half of all collected macrophages (a total of 3 × 106 to 6 x 106 cells) on a cell culture insert with an effective area of 4.2 cm², which is placed on top of the well of dissociated DRG.
   NOTE: During the co-culture period, macrophages are grown in the Neurobasal/B-27 neuron culture medium. Adequate survival of macrophages under this condition was confirmed.

3. Treatment of db-cAMP (dibutyryl-cyclic AMP)  and Collection of Macrophage CM

NOTE: Start db-cAMP treatment 4 h after the neuron-macrophage co-cultures.

1. Add 2 µL of 100 µM db-cAMP solution to the neuron-macrophage co-cultures. Add the same volume of PBS for a control experiment.
2. After 24 h, fill an empty well with 1 mL of macrophage culture medium in the same 6-well plate. Transfer the cell culture insert in the neuron-macrophage co-cultures to the empty well with a macrophage culture medium. Keep the cells under the same condition for 72 hours without changing the medium.
   NOTE: We add only 1 mL of macrophage culture medium during CM collection to make concentrated CM. During the CM collection, if needed, we added more to completely cover the macrophages within the insert.
3. After 72 h, centrifuge the macrophage CM at 239 x g for 5 min to remove the cellular components. Pass the supernatant through a 0.2-µm filter to remove any remaining cellular debris. Store the collected CM at -70 °C until use.