This protocol is supported by a grant NRF-2015R1A2A1A01003410 from the Ministry of Science, ICT and Future Planning, Republic of Korea.

All experiments involving animals were approved by the institutional Animal Care and Use Committee of Ajou University School of Medicine.
1. Culture Preparation of Dissociated Adult DRG Neuron
<ol>
	<li>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&#176;C for 2 h or at 4&#176;C overnight. Then, wash the plate twice with distilled water.</li>
	<li>Incubate the plate with laminin solution at a concentration of 3 &#181;g/mL for 2 h at room temperature, and then wash the plate twice with distilled water. Dry the plate at room temperature at least for 1 h.</li>
	<li>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 DRGs neurons are obtained from adult C57BL6 male mice aged between 8 weeks to 12 weeks old.</li>
	<li>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.</li>
	<li>Store the dissected DRGs in a 60 mm Petri dish containing 5 mL of cold Dulbecco&#39;s Modified Eagle Medium (DMEM) on ice until all the 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 the next step even if there are still remaining DRGs.</li>
	<li>Transfer the DRGs to a 1.5 mL Eppendorf tube using a blue pipette tip (1000 &#181;L) 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 &#176;C incubator. Immobilize the tube on the shaker floor using the tape.</li>
	<li>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.</li>
	<li>Transfer the DRGs to a 15 mL conical tube using a blue pipette tip (1000 &#181;L) with a cut-off end. Then pipette up and down gently at least 15 times using a blue tip (1000 &#181;L) to make a homogeneous cell suspension. Avoid making bubbles and try not to touch the bottom of conical tube with a pipette tip.</li>
	<li>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.</li>
	<li>Pass the cell suspension through a 70-&#181;m 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.</li>
	<li>Plate all collected DRG neurons (a total of approximate 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&#176;C until the macrophage co-cultures.</li>
</ol>
2. Co-culture of P rimary Peritoneal Macrophages on A Cell Culture Insert
NOTE: Establish the co-cultures 4 h after the initial plating of the dissociated DRG neurons
<ol>
	<li>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 a leakage of lavage fluid.</li>
	<li>Puncture the peritoneum using a syringe with a 22G needle and inject 10 mL of ice-cold phosphate-buffered salin (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.</li>
	<li>Centrifuge the lavage fluid at 239 x g for 10 min at 4 &#176;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 &#176;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.</li>
	<li>Resuspend the pelleted cells in 1 mL of Neurobasal/B-27 medium. Plate half of all collected macrophages (a total of 3 &#215; 106 to 6 x 106 cells) on a cell culture insert with the 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 Neurobasal/B-27 neuron culture medium. Adequate survival of macrophages under this condition was confirmed.</li>
</ol>
3. Treatment of Db-Camp and Collection of Macrophage CM
NOTE: Start db-cAMP treatment 4 h after the neuron-macrophage co-cultures.
<ol>
	<li>Add 2 &#181;L of 100 &#181;M db-cAMP solution to the neuron-macrophage co-cultures. Add the same volume of PBS for a control experiment.</li>
	<li>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 macrophage culture medium. Keep the cells under the same condition for 72 h 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.</li>
	<li>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-&#181;m filter to remove any remaining cellular debris. Store the collected CM at -70 &#176;C until use.</li>
</ol>
4. Neurite Outgrowth Assay with Collected CM
<ol>
	<li>Before setting up a separate adult DRG neuron culture, pre-coat an 8-well chamber slide following the same procedure described in 1.1 and 1.2.</li>
	<li>Obtain dissociated adult DRG neurons in Neurobasal medium supplemented with B27 using the same methods described from 1.3 to 1.10. Plate 5 x 104 cells per well onto the pre-coated 8-well chamber slide.</li>
	<li>Place the chamber slide in a 37 &#176;C incubator for 2 h, allowing the cells to attach to the bottom. Then, replace the culture medium with the thawed CM that is preheated at 37 &#176;C. 
	NOTE: Be careful not to touch the bottom of the 8-well chamber slide when removing the culture medium and adding the collected CM.</li>
	<li>15 h after the initial plating, remove the medium and wash the cells with PBS once. Then, add 200 &#181;L of ice-cold 4% paraformaldehyde solution to the wells, and incubate the cells with the paraformaldehyde solution for 20 min at 4 &#176;C 
	NOTE: The DRG neuron culture for neurite outgrowth assay should be precisely restricted to 15 h. Under this condition, there is no appreciable neurite outgrowth.</li>
	<li>Wash three times with ice-cold PBS and then perform blocking with 10% normal goat serum (NGS) with 0.1% Triton-X for 30 min. Incubate the fixed cells with primary antibody (anti-Tuj-1) solution diluted with 10% NGS (at a concentration of 1 &#181;g/mL) for 4 h at room temperature or overnight at 4 &#176;C.</li>
	<li>Wash three times with PBS, and then incubate the cells with secondary antibody (goat-anti mouse) solution for 2 h at room temperature. Then, wash twice with PBS and mount the culture slide with a coverslip (24 &#215; 50 mm) using mounting solution.</li>
	<li>Take images using a fluorescence microscope to visualize neurite outgrowth.</li>
</ol>

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	<li>Donnelly, D. J., Popovich, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+its+role+in+neuroprotection,+axonal+regeneration+and+functional+recovery+after+spinal+cord+injury.">Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury.</a> Exp Neurol. 209 (2), 378-388 (2008).</li><li>Festoff, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minocycline+neuroprotects,+reduces+microgliosis,+and+inhibits+caspase+protease+expression+early+after+spinal+cord+injury.">Minocycline neuroprotects, reduces microgliosis, and inhibits caspase protease expression early after spinal cord injury.</a> J Neurochem. 97 (5), 1314-1326 (2006).</li><li>Bowers, C. A., Kundu, B., Hawryluk, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylprednisolone+for+acute+spinal+cord+injury:+an+increasingly+philosophical+debate.">Methylprednisolone for acute spinal cord injury: an increasingly philosophical debate.</a> Neural Regen Res. 11 (6), 882-885 (2016).</li><li>Gensel, J. C., Kigerl, K. A., Mandrekar-Colucci, S. S., Gaudet, A. D., Popovich, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Achieving+CNS+axon+regeneration+by+manipulating+convergent+neuro-immune+signaling.">Achieving CNS axon regeneration by manipulating convergent neuro-immune signaling.</a> Cell Tissue Res. 349 (1), 201-213 (2012).</li><li>DiSabato, D. J., Quan, N., Godbout, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroinflammation:+the+devil+is+in+the+details.">Neuroinflammation: the devil is in the details.</a> J Neurochem. 139 Suppl 2, 136-153 (2016).</li><li>Jin, X., Yamashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+in+central+nervous+system+repair+after+injury.">Microglia in central nervous system repair after injury.</a> J Biochem. 159 (5), 491-496 (2016).</li><li>Yin, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage-derived+factors+stimulate+optic+nerve+regeneration.">Macrophage-derived factors stimulate optic nerve regeneration.</a> J Neurosci. 23 (6), 2284-2293 (2003).</li><li>Yin, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncomodulin+is+a+macrophage-derived+signal+for+axon+regeneration+in+retinal+ganglion+cells.">Oncomodulin is a macrophage-derived signal for axon regeneration in retinal ganglion cells.</a> Nat Neurosci. 9 (6), 843-852 (2006).</li><li>Gensel, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+promote+axon+regeneration+with+concurrent+neurotoxicity.">Macrophages promote axon regeneration with concurrent neurotoxicity.</a> J Neurosci. 29 (12), 3956-3968 (2009).</li><li>Barrette, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+of+myeloid+cells+for+axon+regeneration.">Requirement of myeloid cells for axon regeneration.</a> J Neurosci. 28 (38), 9363-9376 (2008).</li><li>Kwon, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+macrophages+to+enhanced+regenerative+capacity+of+dorsal+root+ganglia+sensory+neurons+by+conditioning+injury.">Contribution of macrophages to enhanced regenerative capacity of dorsal root ganglia sensory neurons by conditioning injury.</a> J Neurosci. 33 (38), 15095-15108 (2013).</li><li>Niemi, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+for+macrophages+near+axotomized+neuronal+cell+bodies+in+stimulating+nerve+regeneration.">A critical role for macrophages near axotomized neuronal cell bodies in stimulating nerve regeneration.</a> J Neurosci. 33 (41), 16236-16248 (2013).</li><li>Hannila, S. S., Filbin, M. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+the+monocyte+chemokine+CCL2+in+dorsal+root+ganglion+neurons+causes+a+conditioning-like+increase+in+neurite+outgrowth+and+does+so+via+a+STAT3+dependent+mechanism.">Overexpression of the monocyte chemokine CCL2 in dorsal root ganglion neurons causes a conditioning-like increase in neurite outgrowth and does so via a STAT3 dependent mechanism.</a> Exp Neurol. 275 Pt 1, 25-37 (2016).</li><li>Cafferty, W. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioning+injury-induced+spinal+axon+regeneration+fails+in+interleukin-6+knock-out+mice.">Conditioning injury-induced spinal axon regeneration fails in interleukin-6 knock-out mice.</a> J Neurosci. 24 (18), 4432-4443 (2004).</li><li>Cai, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+cyclic+AMP+controls+the+developmental+loss+in+ability+of+axons+to+regenerate.">Neuronal cyclic AMP controls the developmental loss in ability of axons to regenerate.</a> J Neurosci. 21 (13), 4731-4739 (2001).</li><li>Neumann, S., Woolf, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+dorsal+column+fibers+into+and+beyond+the+lesion+site+following+adult+spinal+cord+injury.">Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury.</a> Neuron. 23 (1), 83-91 (1999).</li></ol>

The authors have nothing to disclose.

There are several critical steps for the generation of this co-culture system. It is important to ensure that the mouse DRG neurons and peritoneal macrophages are prepared fresh and healthy. We have experienced diminished neurite outgrowth activity of the CM when the dissection of all the DRGs took more than 30 min. In addition, the contamination of blood components in peritoneal macrophages also led to a decrease of neurite outgrowth activity in the CM. In order to elicit robust neuron-macrophage interaction by db-cAMP,...

A variety of studies have sought to enhance CNS axon regeneration after the injuries of the spinal cord or brain. Inflammatory reactions, inevitably accompanying the injuries in the nervous system, are traditionally thought to participate in secondary pathological processes leading to the deleterious outcomes1,2. Indeed, methylprednisolone that can suppress inflammatory reactions is the only approved therapy for acute spinal cord injury3. However, more recent studies have provided evidence that macrophages, a representative inflammatory cell type, can participate in the regeneration or repair of injured nervous system4,5,6. For example, infiltrating macrophages following a lens injury produce pro-regenerative molecules to promote the regeneration of the retinal ganglion neurons7,8. In addition, transplanted DRG neurons increased axon growth up the region where macrophages were activated by zymosan9. Moreover, the macrophages at the lesion site can create a growth-permissive milieu for injured peripheral nerves10.
Our work also provided strong evidence that macrophages can contribute to the capacity of axon regeneration in adjacent neurons. We have shown that the activation of macrophages in the dorsal root ganglia (DRG) were essential in the enhanced regenerative capacity of DRG sensory neurons following a preconditioning peripheral nerve injury11. Similar research was independently reported from another laboratory12. We also showed that intraganglionic injection of dibutyryl cyclic AMP (db-cAMP), which is a well-known molecule to enhance the capacity of axon regeneration13, induced the activation of macrophages. The deactivation of macrophages abolished the effects of db-cAMP on neurite outgrowth activity. Subsequent works identified injury-induced expression of CCL2 in neurons as a signal to stimulate macrophages with a pro-regenerative phenotype14,15.
Based on the above experimental results, we have established an in vitro model resembling molecular events that occur in the DRGs following a preconditioning injury model11,14. In this model, db-cAMP is applied to the neuron-macrophage co-cultures eliciting intercellular signaling that leads to the activation of macrophages with a pro-regenerative phenotype. Here, we describe detailed protocols by which we can generate macrophages that secrete molecular factors promoting neurite outgrowth (Figure 1). This experimental model illustrates a concept that macrophages can be stimulated or induced to support axon regeneration following the injuries to nervous system. Our model will also be useful in studying mechanisms in intercellular signaling that leads to the activation of pro-regenerative macrophages.

<table><tbody><tr><td>Cell culture insert transparent PET membrane 0.4&amp;mu;m pore size</td><td>Corning,Falcon</td><td>353090</td><td>Transparent PET membrane with 0.4-&amp;mu;m pore size, for 6-well plate</td></tr><tr><td>70-&amp;mu;m nylon cell strainer</td><td>Corning, Falcon</td><td>352350</td><td></td></tr><tr><td>8-well culture slide</td><td>Biocoat</td><td>354632</td><td>with a uniform application of Poly-D-Lysine</td></tr><tr><td>Red blood cell lysis buffer</td><td>Qiagen</td><td>158904</td><td></td></tr><tr><td>Collagenase from &lt;em&gt;Clostridium histolyticum&lt;/em&gt;</td><td>Sigma-Aldrich</td><td>C9407-100MG</td><td></td></tr><tr><td>Neurobasal medium</td><td>Thermo Fisher Scientific, Gibco</td><td>21103-049</td><td>Containing 1% glutamax and 1% penicilin-streptomycin</td></tr><tr><td>B-27 supplement, serum free</td><td>Thermo Fisher Scientific, Gibco</td><td>17504-044</td><td>extracellular solution</td></tr><tr><td>Glutamax</td><td>Thermo Fisher Scientific, Gibco</td><td>35050-061</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Thermo Fisher Scientific, Gibco</td><td>15140-122</td><td></td></tr><tr><td>Poly-D-lysine Hydrobromide</td><td>Sigma-Aldrich</td><td>P6407-5MG</td><td></td></tr><tr><td>Laminin</td><td>Thermo Fisher Scientific, Invitrogen</td><td>23017-015</td><td></td></tr><tr><td>Adenosine 3&#039;, 5&#039;-cyclic monophosphate, N&lt;sup&gt;6&lt;/sup&gt;,O2&#039;-dibutyryl-, sodium salt</td><td>Merck Millipore Corporation, Calbiochem</td><td>28745</td><td></td></tr><tr><td>10% Normal Goat Serum</td><td>Thermo Fisher Scientific</td><td>16210072</td><td></td></tr><tr><td>Triton-X-100</td><td>Daejung Chemical and Metal Co</td><td>8566-4405</td><td></td></tr><tr><td>Anti &amp;beta; III tubulin (Tuj-1)</td><td>Promega Corporation</td><td>G7121</td><td>Mouse monoclonal antibody</td></tr><tr><td>Goat anti-Mouse IgG (H+L) secondary antibody</td><td>Thermo Fisher Scientific, Invitrogen</td><td>A11005</td><td></td></tr><tr><td>Hemacytometer</td><td>Marienfeld-Superior</td><td>N/A</td><td></td></tr><tr><td>Cell culture CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Panasonic</td><td>N/A</td><td></td></tr><tr><td>Dissecting stereomicroscope</td><td>Carl Zeiss</td><td>Stemi DV4</td><td></td></tr><tr><td>Twist shaker</td><td>FINEPCR</td><td>Tw3t</td><td></td></tr><tr><td>Tabletop centrifuge</td><td>Sorvall</td><td>N/A</td><td></td></tr><tr><td>Confocal microscope</td><td>Olympus America Inc</td><td>IX71</td><td></td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>VWR International, Hyclone</td><td>SH30919.03</td><td></td></tr><tr><td>Friedman Pearson Rongeurs</td><td>FST (Fine Science Tools)</td><td>16021-14</td><td>Stainless steel, 14cm, curved, single joint action</td></tr><tr><td>Fine Scissors - Tungsten Carbide &amp;amp; ToughCut</td><td>FST (Fine Science Tools)</td><td>14558-11</td><td>sharp, serrated</td></tr><tr><td>Dumont #7 Forceps</td><td>FST (Fine Science Tools)</td><td>11272-30</td><td>Dumoxel, 0.07 x 0.04mm, curved</td></tr><tr><td>Vannas Spring Scissors</td><td>FST (Fine Science Tools)</td><td>15000-00</td><td>straight, 3mm cutting-edge, sharp</td></tr><tr><td>Qualitron DW-41 Micro-Centrifuge</td><td>Artisan Technology Group</td><td>DW-41</td><td>Input Voltage: 115VAC</td></tr></tbody></table>

neuron-macrophage co-cultures to activate macrophages secreting molecular factors with neurite outgrowth activity

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which macrophages are induced to produce conditioned medium (CM) that promotes neurite outgrowth. Adult dorsal root ganglion (DRG) neurons are acutely dissociated and plated. After the neurons are stably attached, peritoneal macrophages are co-cultured on a cell culture insert overlaid on the same well. Dibutyryl cyclic AMP (db-cAMP) is applied to the co-cultures for 24 h, after which the cell culture insert containing the macrophages is moved to another well to collect CM for 72 h. The CM from the co-cultures treated with db-cAMP, when applied to a separate adult DRG neuron culture, exhibits robust neurite outgrowth activity. The CM obtained from the db-cAMP-treated cultures consisting of single cell type alone, either DRG neuron or peritoneal macrophage, did not exhibit neurite outgrowth activity. This indicates that the interaction between neurons and macrophages is indispensable for the activation of macrophages secreting molecular factors with neurite outgrowth activity into CM. Thus, our co-culture paradigm will also be useful to study intercellular signaling in the neuron-macrophage interaction to stimulate the macrophages to be endowed with a pro-regenerative phenotype.

We describe a protocol that can generate macrophages capable of secreting molecular factors with neurite outgrowth activity. The macrophage CM obtained from the co-cultures treated with db-cAMP resulted in robust neurite outgrowth when applied to a separate DRG neuron culture (Figure 2A). In comparison, CM obtained from the PBS-treated co-cultures did not induce neurite outgrowth in our 15-h culture duration (Figure 2B). When db-...

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Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity

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.

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which ...

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Neuroscience

8.7K Views.  Ajou University School of Medicine. 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.

Video: Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

Cancer Research

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Immunology and Infection

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Biology

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

Developmental Biology

Co-Culturing Microglia and Cortical Neurons Differentiated from Human Induced Pluripotent Stem Cells

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia in health and disease. Furthermore, iPSC-derived microglia can be maintained in co-culture with iPSC-derived cortical neurons, which enable investigations of microglia-neuron interactions that are hypothesized to be dysregulated in a number of neuropsychiatric disorders. Human iPSCs were differentiated to generate microglia using an adapted version of a protocol developed by the Fossati group, and the iPSC-derived microglia were validated with marker analysis and real-time PCR. Human microglia generated using this protocol were positive for the markers CD11C, IBA1, P2RY12, and TMEM119, and expressed the microglial-related genes AIF1, CX3CR1, ITGAM, ITGAX, P2RY12, and TMEM119. Human iPSC-derived cortical neurons that had been differentiated for 30 days were plated with microglia and maintained in co-culture until day 60, when experiments were undertaken. The density of dendritic spines in cortical neurons in co-culture with microglia was quantified under baseline conditions and in the presence of pro-inflammatory cytokines. In order to examine how microglia modulate neuronal function, calcium imaging experiments of the cortical neurons were undertaken using the calcium indicator Fluo-4 AM. Live calcium activity of cortical neurons was obtained using a confocal microscope, and fluorescence intensity was quantified using ImageJ. This report describes how co-culturing human iPSC-derived microglia and cortical neurons provide new approaches to interrogate the effects of microglia on cortical neurons.

This protocol describes a methodology to differentiate microglia from human iPSCs and maintain them in co-culture with iPSC-derived cortical neurons in order to study mechanistic underpinnings of neuroimmune interactions using human neurons and microglia.

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

 Primary Microglia and Neuron Co-Culture Protocol for CNS Interaction Studies

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting&#160;reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

Author Spotlight: In Vitro Co-Culture Model for Studying Microglia-Neuronal Interactions in Disease Conditions

This protocol describes a microglia-neuronal co-culture established from primary neuronal cells isolated from mouse embryos at embryonic days 15-16 and primary microglia generated from the brains of neonatal mice at post-natal days 1-2.

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS ...

Developing a Neuron and Macrophage Coculture In Vitro

Source:Yun, H. J., et al. <a href="https://app.jove.com/v/56920">Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity.</a> J. Vis. Exp. (2018).
This video shows the co-culturing of murine dorsal root ganglion neurons and macrophages. Neurons are seeded on a coated plate, while macrophages are collected from an euthanized mouse, treated, and added to the culture insert. Treatment with db-cAMP activates neurons and prompts macrophages to adopt a pro-regenerative phenotype to support neuronal outgrowth.

Opracowanie kokultury neuronów i makrofagów in vitro

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

	Before setting up the culture, pre-coat a 6-well plate with ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Co-culture and Interaction Studies

Using a Macrophage Conditioned Medium to Promote Neuron Extensions

Source: Yun, H. J., et al. <a href="https://app.jove.com/v/56920">Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity.</a> J. Vis. Exp. (2018).
This video demonstrates an assay for promoting the growth of neuron projections known as neurite outgrowth using the macrophage-conditioned medium. Co-culture neurons and macrophages with cAMP and harvest the macrophage-derived conditioned medium. Growing the neurons with the conditioned medium promotes neurite outgrowths. These outgrowths are fluorescently labeled and visualized using a fluorescence microscope.

Używanie pożywki kondycjonowanej makrofagami w celu promowania rozszerzeń neuronów

1. Treatment of Db-Camp and Collection of Macrophage Conditioned Medium
NOTE: Start db-cAMP treatment 4 h after the neuron-macrophage co-cultures.

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