The current protocol presents experimental procedures to stimulate cultured macrophages to be endowed with capacity to release molecular factors that promote neurite outgrowth. Treatment of cAMP to the neuron-macrophage co-cultures induces the macrophages to produce conditioned medium that possesses strong neurite outgrowth activity.
The overall goal of this procedure is to generate macrophages that can secrete molecular factors promoting neurite growth. This method can help answer key questions on how neurons and macrophages interact with each other to activate the pro-regenerative macrophagicphenotype to promote excellent regeneration. The main advantage of this technique is that we use Cyclic AMP, an adenosine serum molecule, which is much more physiologic enzymesone used in previous studies to stimulate macrophages with the pro-regenerative phenotype.
We first had the idea for this method when we found that macrophage activation is essential in the enhancement of axion growth capacity and those can produce excellent neurons following precaution in permanent lobe injury. Before setting up the culture, precode a six well plate with poly-D Lycine and laminin. Next, incubate the six well plate with 0.01 poly-D Lycine at 37 degrees Celsius for two hours or at four degrees Celsius overnight.
After that, wash the plate twice with distilled water. Then incubate the plate with laminin solution at a concentration of three micrograms per milliliter for two hours at room temperature. Following this, wash the plate twice with distilled water and dry the plate at room temperature for at least one hour.
Now, incise the skin overlying the vertebral column of a euthanized mouse with a surgical blade and dissect the pair of vertebral muscles bilaterally to expose the vertebral bones. Remove the vertebral bones meticulously using a narrow-tipped surgical rongeur until the DRG are fully exposed. Under a dissecting microscope, remove the DRG bilaterally from S1 all the way up to the C1 level using iridectomy scissors and fine tipped forceps.
Transfer the DRG to a 1.5 milliliter eppendorf tube using a blue pipette tip with a cut off end. Then remove DMEM after a quick spin for several seconds using a minicentrifuge. Add one milliliter of DMEM containing 125 units per milliliter type 11 collagenous and incubate the tube for 90 minutes with a gentle rotation using a twister shaker at 37 degrees Celsius.
Afterward, discard collagenase containing DMEM and add one milliliter of fresh DMEM. Transfer the DRG to a 15 milliliter conical tube using a blue pipette tip with a cut off end. Then pipette up and down gently at least 15 times to make a homogenous cell suspension.
Centrifuge the tube at 239 G for three minutes and carefully discard the supernatant with floating debris. Add one milliliter of neurobasil medium supplemented with B27 and re-suspend the cell pellet by gently pipetting up and down five to ten times. Next pass the cell suspension through a 70 micrometer cell strainer overlayed on top of a 50 milliliter conical tube.
Then plate all the collected DRG neurons onto two wells of the six well plate. To prepare primary peritoneal macrophages from an adult mouse, incise the abdominal skin delicately to expose the peritoneum and avoid cutting the peritoneum to prevent a leakage of lavage fluid. Next puncture the peritoneum using a syringe with a 22 gauge needle and inject 10 milliliters of ice cold PBS into the peritoneal cavity.
Gently massage the peritoneum for one to two minutes. Then pull out the needle and squeeze out the PBS through the needle puncture site and collect the lavage fluid in a 50 milliliter conical tube. Afterward centrifuge the lavage fluid at 239 g for 10 minutes at four degrees Celsius to pellet the cellular components.
Subsequently re-suspend the pellet with three milliliters of the red blood cell lysis buffer for three minutes at room temperature. Then centrifuge the cell suspension again at 239 g for 10 minutes at four degrees Celsius. Re-suspend the pelletted cells in one milliliter of neurobasil B27 medium.
Plate half of all collected macrophages on a cell culture insert with the effective area of 4.2 centimeters squared, which is placed on top of the well of dissociated DRG. Four hours after the neuron macrophage have been co-cultured add two microliters of 100 micromolar DB cyclic amp solution to the neuron macrophage co-cultures. After 24 hours, fill an empty well with one milliliter of macrophage culture medium in the same six well plate.
Transfer the cell culture insert in the neuron macrophage co-culture to the empty well with macrophage culture medium. After 72 hours, centrifuge the macrophage conditioned medium at 239 times g for five minutes to remove the cellular components. Pass the supernatant through a 0.2 micrometer filter to remove any remaining cellular debris.
In this procedure, precoat an eight well chamber slide with poly-D lycine and laminin. Then obtain the dissociated adult DRG neurons and neurobasil medium, supplemented with B27 using the same methods as described previously. Plate five times 10 to the four cells per well onto a precoated eight well chamber slide.
Next place the chamber slide in a 37 degree Celsius incubator for two hours allowing the cells to attach to the bottom. Then replace the culture medium with the thawed, conditioned medium that is preheated at 37 degrees Celsius. 15 hours after the initial plating, remove the medium and wash the cells with PBS once, then add 200 microliters of ice cold, 4%paraformaldehyde solution to the wells and incubate the cells with the paraformaldehyde solution for 20 minutes at four degrees Celsius.
Subsequently, incubate the fixed cells with primary antibody solution diluted with 10%normal goat serum for four hours at room temperature or overnight at four degrees Celsius. Afterward, take images using a fluorescence microscope to visualize neurite outgrowth. The macrophage conditioned medium obtained from the co-cultures treated with DB cyclic AMP resulted in robust neurite outgrowth when applied to a separate DRG neuron culture.
In comparison, conditioned medium obtained from the PBS treated co-cultures did not induce neurite outgrowth. When DB cyclic AMP is treated to the culture with macrophage alone, conditioned medium was not effective in supporting neurite outgrowth. This suggests that neuron macrophage interaction is indispensable to stimulate macrophages to be endowed with a pro-regenerative capacity.
We also tested the conditioned medium obtained from DB cyclic AMP treated neuron only culture and found no neurite outgrowth. It is important to remember that neurons and microplates should be prepared fresh and healthy. We experienced diminished neurite outgrowth activity of the constant medium when dissection lasted too long or peritoneum macrophages were contaminated with blood components.
In this protocol, the dissociated DRG neurons were fiscally separated from the macrophages on the cell culture insert. Therefore our co-culture system will allow the identification of cellular factors responsible for intercellular signaling that mediate the activation of fully regenerative macrophages.
