Name: neuron-macrophage co-cultures to activate macrophages secreting molecular factors with neurite outgrowth activity
Uploaded: 2018-03-30T15:00:00.000+00:00
Duration: 8 min 52 s
Description: Watch this Scientific Journal Video about Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity at JoVE.com

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>

<ol>
	<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. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+cyclic+AMP+signaling+in+promoting+axonal+regeneration+after+spinal+cord+injury.">The role of cyclic AMP signaling in promoting axonal regeneration after spinal cord injury.</a> Exp Neurol. 209 (2), 321-332 (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=CCL2+Mediates+Neuron-Macrophage+Interactions+to+Drive+Proregenerative+Macrophage+Activation+Following+Preconditioning+Injury.">CCL2 Mediates Neuron-Macrophage Interactions to Drive Proregenerative Macrophage Activation Following Preconditioning Injury.</a> J Neurosci. 35 (48), 15934-15947 (2015).</li><li>Niemi, J. P., DeFrancesco-Lisowitz, A., Cregg, J. M., Howarth, M., Zigmond, R. 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, thorough washing would also be a critical step to ensure complete removal of tissue debris and elimination of potentially remaining cytotoxic components, such as collagenase or RBC lysis buffer. Another point to be reiterated is that the roots attached to DRGs should be removed as much as possible. Remaining stubs of roots would increase the contamination of Schwann cells in DRG neuron culture. Schwann cells can produce high amount of neurotrophic factors in culture that might confuse the results.
In this protocol, dissociated macrophages were plated on a cell culture insert separated from the DRG neurons plated on the bottom of the well plate. Our previous study did not show significant difference in the extent of neurite outgrowth activity between the CMs collected from direct co-cultures (allowing physical contacts between the two cell types) and the co-cultures with the two cell types separated by a cell culture insert11. This result indicated that neurons and macrophages are communicating with each other via soluble molecules released from either cell type, not by direct physical contacts. It was shown that CCL2, secreted from DRG neurons, is responsible for activating macrophages into a pro-regenerative phenotype in this co-culture model14. Thus, our co-culture system will allow the elucidation of more detailed intercellular signaling that mediates the activation of pro-regenerative macrophages.
The culture was precisely restricted to 15 h during the neurite outgrowth assay in this protocol. In pilot experiments, we examined several different culture time and found that with a minimal extent of neurite outgrowth in control condition (15 h), highly robust neurite outgrowth was achieved with the CM treated with db-cAMP. Mouse DRG neurons, if not preconditioned, do not grow any significant neurites within this time frame. If DRG neurons are cultured longer than 15 h, however, they start to show some degree of neurite outgrowth even in control, not preconditioned condition, which would obscure any effect of the db-cAMP-treated CM on neurite outgrowth. Similar result was reported in the neurite outgrowth assay using rat DRG neurons in a previous study16.
Some previous studies used zymosan, a yeast cell wall preparation, to activate macrophages with a pro-regenerative phenotype7,9. However, the macrophages stimulated by zymosan released not only pro-regenerative molecules but also cytotoxic factors. When the protein components in macrophage CM treated with zymosan were separated by gel-filtration chromatography, macrophage-derived factors &#8805; 30 kDa were cytotoxic while factors &#8804; 30 kDa promoted axon regeneration7. In addition, zymosan injection to the spinal cord can lead to overt death of neurons and axons9. In comparison, our protocol to generate pro-regenerative macrophages has shown to be more physiologic than the previous one using zymosan. In fact, db-cAMP injection to the DRG neurons enhanced their capacity to growth axons following an injury to the central branch without any report on cellular damages17,18. Throughout a series of our experiments, we have never observed any significant decrease of the DRG neuron density in the culture following application of the db-cAMP-treated CM. We speculate that our protocol allows us to produce solely pro-regenerative macrophages without concurrent neurotoxicity. Therefore, the protocol and experimental model reported here would be useful to identify molecular signatures of pro-regenerative macrophages and to understand by what mechanisms the pro-regenerative phenotype is evolved.

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-cAMP is treated to the culture with macrophage alone, CM obtained from this condition was not effective in supporting neurite outgrowth (Figure 2C). This suggests that neuron-macrophage interaction is indispensible to stimulate macrophages to be endowed with a proregenerative capacity. We also tested if CM obtained from db-cAMP-treated neuron-only culture and found no neurite outgrowth (Figure 2D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56920/56920fig1.jpg" /> 
Figure 1: A schematic diagram illustrating procedures to obtain conditioned medium containing neurite outgrowth activity. Adult DRG neurons (2 x 106 cells per each well) are cultured for 4 h. Then, peritoneal macrophages are cultured in insert well, located above the well in which DRG neurons were cultured. 4 h after plating, either PBS or dibutyryl cAMP is treated. After 24 h incubation, the insert well is transferred to another well filled with about 1ml medium. Then, the transferred well is incubated for 72 h. Then, the incubated conditioned medium (CM) is collected. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56920/56920fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56920/56920fig2.jpg" /> 
Figure 2: Representative results of neurite outgrowth assay using conditioned medium obtained from various conditions. (A, B) Adult DRG neurons were acutely dissociated and cultured for exactly 15 h. 2 h after plating, culture medium was replaced with conditioned medium (CM) obtained from neuron-macrophage co-cultures treated with either PBS (A) or dibutyryl cAMP (db-cAMP) (B). Only the CM treated with db-cAMP exhibited robust neurite outgrowth activity. (C, D) CM obtained from cultures consisting of either macrophage (C) or neuron (D) alone that was treated with db-cAMP did not support neurite outgrowth. Scale bars indicate 200 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56920/56920fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity at JoVE.com

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 ...

neuron-macrophage-co-cultures-to-activate-macrophages-secreting

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.7K Views.  Ajou University School of Medicine. Het huidige protocol presenteert experimentele procedures ter stimulering van gekweekte macrofagen te worden begiftigd met capaciteit om moleculaire factoren ter bevordering van de uitgroei van de neurite vrij te geven. Behandeling van kamp tot de neuron-macrofaag co culturen induceert de macrofagen produceren geconditioneerde medium dat sterke neurite uitgroei activiteit bezit.

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

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

Cancer Research

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

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 ...

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g., beta-amyloid clearance in Alzheimer&#8217;s disease) to development of the healthy brain (e.g., synaptic pruning)1-6. The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system7. While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g., astrocytes) and the rest of the body (e.g., peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g. ,brain injury/disease).

Microglia are the resident immune cells of the central nervous system (CNS) with a high capacity to phagocytose or engulf material in their extracellular environment. Here, a broadly applicable, reliable, and highly quantitative assay for visualizing and measuring microglia-mediated engulfment of synaptic components is described.

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. Microglia are mesodermal cells that function similarly to macrophages in surveying inflammatory stress. The classical (M1-type) and alternative (M2-type) activations of macrophages have also been extended to microglia in an effort to better understand the underlying interplay these phenotypes have in neuroinflammatory conditions such as Parkinson&#39;s, Alzheimer&#39;s, and Huntington&#39;s Diseases. In vitro experimentation utilizing primary microglia offers rapid and reliable results that may be extended to the in vivo environment. Although this is a clear advantage over in vivo experimentation, isolating microglia while achieving adequate yields of optimal purity has been a challenge. Common methods currently in use either suffer from low recovery, low purity, or both. Herein, we demonstrate a refinement of the column-free CD11b magnetic separation method that achieves a high cell recovery and enhanced purity in half the amount of time. We propose this optimized method as a highly useful model of primary microglial isolation for the purposes of studying neuroinflammation and neurodegeneration.

Here, we present a protocol to isolate microglia from postnatal mouse pups (day 1) for in vitro experimentation. This improvised method of isolation generates both high yield and purity, a significant advantage over alternate methods that allows broad range experimentation for the purposes of elucidating microglial biology.

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. ...

A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development and implementation of biomedical devices and tissue engineering constructs. Macrophages, an innate immune cell, are key players in the foreign body reaction because they remain at the implant site for the lifetime of the device, and are commonly studied to gain an understanding of this detrimental host response. Many biomaterials researchers have shown that adsorbed protein layers on implanted materials influence macrophage behavior, and subsequently impact the host response. The methods in this paper describe an in vitro model using adsorbed protein layers containing cellular damage molecules on polymer biomaterial surfaces to assess macrophage responses. An NF-&#1082;B/AP-1 reporter macrophage cell line and the associated colorimetric alkaline phosphatase assay were used as a rapid method to indirectly examine NF-&#1082;B/AP-1 transcription factor activity in response to complex adsorbed protein layers containing blood proteins and damage-associated molecular patterns, as a model of the complex adsorbed protein layers formed on biomaterial surfaces in vivo.

This protocol provides researchers with a rapid, indirect method of measuring TLR-dependent NF-&#1082;B/AP-1 transcription factor activity in a murine macrophage cell line in response to a variety of polymeric surfaces and adsorbed protein layers that model the biomaterial implant microenvironment.

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development ...

Bioengineering

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

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 ...

Biology

 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.remotexs.ntu.edu.sg/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.

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 ...

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

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