JP acknowledges funding support from the Natural Sciences and Engineering Research Council of Canada and the University of Saskatchewan College of Medicine. YD acknowledges funding support from the University of Saskatchewan College of Medicine Startup Fund, the Natural Sciences and Engineering Research Council of Canada Discovery Grant (RGPIN-2023-03659), MS Canada Catalyst Grant (1019973), Saskatchewan Health Research Foundation Establishment Grant (6368), and Brain Canada Foundation Future Leaders in Canadian Brain Research Grant. Figure 1A, Figure 2A, and Figure 3A were created with BioRender.com.

All animals used in this study were housed and handled with approval from the University Animal Care Committee (UACC) of the University of Saskatchewan and the Canadian Council on Animal Care (CCAC). Post-natal days 0-2 CD1 male and female mice and&#160;embryonic days&#160;16-18 (E16-18) embryos from pregnant CD1 mice were used for this study. The details of the reagents and the equipment used are listed in the Table of Materials.
1. Primary microglia culture
NOTE: It is crucial to time the mixed glia and neuronal cultures so that microglia are mature and ready for harvest within 2 days after neurons are seeded into a 96-well plate.
<ol>
	<li>Preparation
	<ol>
		<li>Pre-warm complete glia culture media, which includes Dulbecco&#39;s Modified Eagle Medium (DMEM) with high glucose supplemented with 10% heat-inactivated fetal bovine serum, 50 U/mL Penicillin/Streptomycin with 2.92 mg/mL of L-glutamine, 1mM sodium pyruvate, 1x non-essential amino acids, and 20 mM of HEPES in a 37 &#176;C water bath.</li>
		<li>Add 5 mL of 10 &#181;g/mL poly-L-ornithine (PO) hydrobromide to each T-75 flask. Gently swirl the flask to ensure that PO completely covers the bottom of the flask. Incubate at 37 &#176;C in a 5% CO2 incubator for at least 1 h. 
		NOTE: This coating step is required to facilitate cell adhesion to the T-75 flasks.</li>
	</ol>
	</li>
	<li>Isolation of brains from P0-P2 neonatal mice 
	NOTE: Sterilize all dissection tools and maintain the sterility of reagents. Ensure adherence to aseptic techniques throughout.
	<ol>
		<li>Before starting dissection, spray down the countertop with 70% ethanol or disinfecting spray where dissection will take place. Set up the dissection microscope and place the Petri dish under the microscope. Pour 20 mL of Leibovitz&#39;s L-15 media into the petri dish. Keep the lid of the Petri dish on while the microscope is not in use to reduce contamination.</li>
		<li>Place down several layers of paper towel and thoroughly spray the paper towels with 70% ethanol. Prepare the dissection tools (dissection scissors, a pair of micro-forceps, and tissue forceps) by thoroughly spraying them with 70% ethanol and laying the tools down on the paper towels.</li>
		<li>Transfer post-natal days 0-2 mouse onto the paper towels and thoroughly spray the mouse with 70% ethanol. Decapitate the mouse using dissection scissors (following institutionally approved protocols).</li>
		<li>While gently holding the head, create a midline incision in the cranium starting from the neck to the nose. Peel the skull back with tissue forceps to reveal the brain.</li>
		<li>Using tissue forceps, gently scoop the brain out by inserting the forceps under the brain and lift the brain out from the head. Immediately place the brain into the&#160;Petri dish containing 20 mL of Leibovitz&#39;s L-15 media.</li>
		<li>Under a dissection microscope, carefully remove the brain stem and the meninges using a pair of micro-forceps. Collect the brains in a 50 mL conical tube containing 5 mL of Leibovitz&#39;s L-15 media.</li>
	</ol>
	</li>
	<li>Brain dissociation, seeding and maintaining mixed glia culture 
	NOTE: Perform all subsequent steps in a biosafety cabinet.
	<ol>
		<li>Mince the brains into approximately 1 mm2 pieces with a sterile disposable scalpel blade. Carefully transfer the minced brains into a new sterile 50 mL tube.</li>
		<li>To the minced brains, add a final concentration of 0.25% trypsin and incubate the tissue in a 37 &#176;C water bath for 20 min. Every 2-3 min, mix the solution by gently inverting the tube to aid tissue dissociation.</li>
		<li>From the 5% CO2 incubator, transfer the T-75 flasks containing 5 mL of PO into the biosafety cabinet. Discard the PO and rinse the flasks 3 times with sterile 1x phosphate buffer solution (PBS). Ensure to thoroughly rinse the flasks as excess PO is toxic to cells.</li>
		<li>Transfer the digested tissue from the 37 &#176;C water bath into the biosafety cabinet. Add 5 mL of complete glia culture media to neutralize the trypsin. Gently mix the tissue suspension using a 10 mL pipette.</li>
		<li>Place a sterile 70 &#181;m nylon mesh cell strainer onto a sterile 50 mL conical tube and wet the mesh by pouring roughly 5 mL of L-15. Carefully decant the cell suspension through the cell strainer. Remove the plunger from a sterile 1 mL syringe, grind the tissue into the mesh using the plunger to get rid of tissue clumps, and generate a homogenized cell suspension.</li>
		<li>Periodically, pour approximately 5 mL of Leibovitz&#39;s L-15 media into the 70 &#181;m nylon mesh and continue grinding the tissue on the mesh with the 1 mL syringe plunger. When little to no visible tissue remains on the mesh, discard the mesh and set the cell suspension aside.</li>
		<li>Mix the cell suspension by gently pipetting up and down. To each flask, add equal volumes of cell suspension containing 1-2 brains with glia culture media to a final volume of 15 mL.</li>
		<li>Incubate the cells at 37 &#176;C in a 5% CO2 incubator overnight. The following day, wash the flasks twice with sterile 1x PBS to remove unattached cells and debris.</li>
		<li>Add 15 mL of fresh complete glia culture media to the culture and incubate at 37 &#176;C in a 5% CO2 incubator. After 3 days, change half the media with 10 mL of fresh glia culture media supplemented with 40 ng/mL of macrophage colony-stimulating factor (MCSF). 
		NOTE: Thereafter, half the media is replaced with 10 mL of fresh glia culture media supplemented with 40 ng/mL of MCSF every 3 days. The culture is maintained for 8-10 days before microglia are harvested.</li>
	</ol>
	</li>
</ol>
2. Primary neuron culture
<ol>
	<li>Preparation
	<ol>
		<li>Thaw B-27 plus supplement from B-27 plus neuronal culture system. Pre-warm neurobasal plus medium from B-27 plus neuronal culture system, 1x Hank&#39;s Balanced Salt Solution (HBSS), and heat-inactivated fetal bovine serum (FBS) at 37 &#176;C in a water bath.</li>
		<li>Coat T-25 flasks with 5 mL of 10 &#181;g/mL PO and incubate the flasks for at least 1 hour at 37 &#176;C. Prepare instruments and supplies for dissection. 
		NOTE: This coating step facilitates cell adhesion to the T-25 flasks.</li>
	</ol>
	</li>
	<li>Brain isolation from E16-E18 mouse embryos 
	NOTE: Sterilize all dissection tools and maintain the sterility of reagents. Ensure adherence to aseptic techniques throughout.
	<ol>
		<li>Before starting dissection, spray down the countertop with 70% ethanol or disinfecting spray where dissection will take place. Set up the dissection microscope and place the Petri dish under the microscope. Pour 20 mL of 1x HBSS into the Petri dish. Keep the lid of the Petri dish on while the microscope is not in use to reduce contamination.</li>
		<li>Place down several layers of paper towel and thoroughly spray the paper towels with 70% ethanol. Prepare the dissection tools (spring scissors, a pair of micro-forceps, and tissue forceps) by thoroughly spraying them with 70% ethanol and laying the tools down on the paper towels.</li>
		<li>Anesthetize a pregnant mouse between gestation days 16-18 using isoflurane and euthanize the mouse by cervical dislocation (following institutionally approved protocols).</li>
		<li>Lay the mouse on its back on a piece of paper towel soaked in 70% ethanol. Disinfect the abdominal area with 70% ethanol. Lift the skin of the lower abdomen with tissue forceps and perform a V-shaped incision from this point to the lower rib cage with dissection scissors.</li>
		<li>Grab the uterine horns containing the embryos with tissue forceps and gently remove the embryos from the abdominal cavity. Briefly disinfect the uterine horns containing embryos with 70% ethanol.</li>
		<li>Dissect one embryo from its individual sac at a time. Using spring scissors, create a midline incision at the top of the skull, starting from the back of the neck to the nose. Then, lift the skull away from the incision site to reveal the brain. Gently scoop out the brain with tissue forceps and transfer the brain to a 10 cm Petri dish containing 15 mL of 1x HBSS.</li>
		<li>Under a dissection microscope, carefully remove the brainstem and the meninges on both sides of the brain cortices with a pair of micro-forceps. Transfer the brain cortices into a 50 mL conical tube containing 10 mL of 1x HBSS and place on ice.</li>
	</ol>
	</li>
	<li>Brain dissociation and seeding of cortical neurons in cell culture 
	NOTE: Perform all subsequent steps in a biosafety cabinet.
	<ol>
		<li>Mince the brains into approximately 1 mm2 pieces in the 50 mL conical tube with a sterile disposable scalpel blade. Transfer the minced brains into a new sterile 50 mL conical tube.</li>
		<li>Add trypsin to the tube with a final concentration of 0.25%. Immerse the tube in a 35-38 &#176;C hot water bath for 15 min and gently mix the tube to homogenize the tissue every 2-3 min.</li>
		<li>Take the tube out of the hot water bath. Gently pipette the tissue suspension up and down to further dissociate cells, then add 0.5 mL of heat-inactivated FBS to the homogenized suspension to neutralize trypsin activity. Avoid air bubbles to minimize toxicity to neuronal cells.</li>
		<li>Place a sterile 70 &#181;m nylon mesh cell strainer onto a sterile 50 mL conical tube and wet the mesh by adding roughly 5 mL of neurobasal media. Filter the cell suspension through the 70 &#181;m nylon mesh cell strainer. Using a 1 mL syringe plunger, gently grind the tissue into the cell strainer. Periodically pour approximately 5 mL of 1x HBSS while continuing grinding to wash the cell strainer.</li>
		<li>Centrifuge the conical tube at 300 x g for 5 min at 4 &#176;C. During this time, dilute B-27 plus supplement in neurobasal plus media to 1x final concentration to use as complete neurobasal media.</li>
		<li>Discard the PO in the T-25 flasks and rinse the flasks three times with 5 mL of 1x PBS.</li>
		<li>Carefully decant to remove the supernatant and resuspend the cell pellet by gently pipetting up and down 2-3 mL of complete neurobasal media. Determine the cell concentration and total cell numbers with a hemocytometer and trypan blue. Expect up to 1.0 x 107 cells per embryo.</li>
		<li>Plate the cells in the PO-coated T-25 flasks with approximately 2.0 x 107 cells per flask in 5 ml of complete neurobasal media. Incubate the cell culture at 37 &#176;C in a 5% CO2 incubator.</li>
		<li>Perform a half media change every 2-3 days by replacing 2.5 mL of media from the culture flask with freshly made complete neurobasal media.</li>
	</ol>
	</li>
</ol>
3. Co-culture of primary neurons and microglia
NOTE: All subsequent steps are to be done in a sterile biosafety cabinet.
<ol>
	<li>Seeding neurons in 96-well plates
	<ol>
		<li>Add 100 &#181;L of 10 &#181;g/mL PO per well and incubate the flasks for at least 1 h at 37 &#176;C to coat the wells with PO. Wash the plate with 1x PBS 3 times before seeding cells and aspirate any remaining liquid after the last wash.</li>
		<li>Neurons grown in T-25 flasks are harvested for co-culture after 2-5 days in culture. Replace the media with 5 mL of 1x Versene solution with 0.25% trypsin and 1 mg/mL DNase I to detach neurons from the flask. Place the flasks inside the incubator for 5-6 min for digestion and gently swirl the flask every 2-3 minutes to aid cell detachment.</li>
		<li>Take the flask out of the incubator and check if cells are detached under a microscope. Add 0.5 mL of heat-inactivated FBS to neutralize the trypsin and DNase I. Transfer cells to a 15 mL conical tube. Wash the flask with 5 mL of complete neurobasal media once to maximize collected neurons. Collect all cells in the same 15 mL conical tube.</li>
		<li>Centrifuge the cell suspension at 300 x g for 5 min at 4 &#176;C. Discard the supernatant and gently resuspend the cell pellet in 2-3 mL of complete neurobasal media.</li>
		<li>Count cells with a hemocytometer and add complete neurobasal media to achieve a final concentration of 7.5 x 105 neurons/mL. Seed 100 &#181;L of the cell suspension to each well in the PO-coated 96-well plate to obtain 7.5 x 104 neurons/well. A yield of 5-6 million neurons per flask is expected.</li>
		<li>Incubate the neurons in a 96-well plate overnight. Add 100 &#181;L of complete neurobasal media to each well the next day. 
		&#8203;NOTE: Once neurons display appropriate neurite process growth (around day 3-4 after seeding in 96-well plates), they are ready for co-culture with microglia.</li>
	</ol>
	</li>
	<li>Isolation and microglia-neuronal co-culture
	<ol>
		<li>Between 8-10 days of mixed glia cultivation, collect microglia by gently washing the T-75 flasks with glia culture media in each flask with a 10 mL pipette, and transfer the cells and media into a sterile 50 mL conical tube. Wash the flasks again with 10 mL fresh glia culture media to detach additional microglia from the flasks and collect the media. A yield of 7.5 x 105 microglia per flask is expected.</li>
		<li>Add 20 mL of fresh glia culture media supplemented with 20 ng/mL MCSF to each flask. Microglia may be harvested once or twice more if the mixed glia culture is maintained.</li>
		<li>Centrifuge the cell collection at 300 x g for 5 min at 4 &#176;C. Carefully remove the supernatant and gently resuspend the pellet in 2 mL of complete neurobasal media.</li>
		<li>Count the cells with a hemocytometer and trypan blue. Add complete neurobasal media to a final concentration of 2.5 x 105 cells/mL.</li>
		<li>From the 96-well neuronal culture plate, remove 100 &#181;L of neurobasal media and add 100 &#181;L of 2.5 x 105 cells/mL cell suspension to seed 2.5 x 104 microglia per well of 7.5 x 104 neurons. Incubate the culture overnight at 37 &#176;C in a 5% CO2 incubator to allow microglia to settle. The co-culture may now be used for downstream experiments.</li>
	</ol>
	</li>
</ol>

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

The authors declare no conflicts of interest.

This article describes a protocol for isolating and culturing mouse primary neurons and primary microglia, which are subsequently used to establish a microglia-neuronal co-culture that can be used to study how microglia and neuron interactions regulate their cellular health and function. This relatively simple and accessible approach can provide critical insights into the mechanisms and functional outcomes of microglia neuron interactions in the CNS.
To achieve an optimal co-culture, several critical aspects in this protocol require extra care. During brain isolations (steps 1.2.6 and 2.2.7), meninges attached to the surface of both the embryonic and post-natal brains should be removed completely before collecting the tissues. Like other neuroglia protocols, incomplete removal of the meninges may result in cell culture becoming contaminated with endothelial cells or blood-derived cells40,41,42. Additionally, when culturing primary neurons (steps 2.3.7 and 3.1.4), it is crucial to generate a single-cell suspension and avoid cell clumps before seeding neurons into flasks or 96-well plates. Neuronal aggregates will not properly adhere in culture and will lead to excessive neuronal stress and cell death. In addition, extended treatment of neurons with trypsin when attempting to dissociate them from T-25 flasks may cause unwanted cell death (step 3.1.2). To mitigate this, neurons may be directly seeded in 96-well plates at the desired cell density following dissociation from embryonic brains (step 2.3.7). Moreover, in this protocol, a 3:1 neuron: microglia ratio was selected to partially represent an elevated amount of microglia that may be present in the CNS microenvironment during neuroinflammation.&#160;Alternatively, the ratio of cells in the co-culture may be adjusted per experimental need. In addition, the timepoint selected for co-culture neurons may be extended if one aims to assess more mature neuronal states.
Notably, when dissociating neurons from T-25 flasks to 96-well plates, using trypsin alone may result in incomplete dissociation and the formation of cell clumps instead of a monolayer culture in 96-well plates (Figure 2D). To address this issue, neurons can be dissociated from the T-25 flask using 1x Versene solution containing 1 mg/mL DNase I. Other factors contributing to neuronal aggregation and reduced viability include incomplete trituration of the cell suspension, inadequate neutralization of trypsin with heat-inactivated FBS, excessive centrifugation, or prolonged cell pellet formation. Therefore, ensuring proper tissue dissociation and the generation of a single-cell suspension are critical for establishing a healthy microglia and neuron co-culture.
This protocol aims to recapitulate microglia-neuronal interactions in vitro to better understand the functional importance of their interactions in the CNS. While other types of co-culture systems have been developed, such as culturing individual cell populations on different surfaces to allow for independent treatment of two cell populations43,44, this mixed culture system enables direct and close contact of microglia and neurons. In addition, when maintained properly through half media changes every 3 days, this mixed culture system can remain healthy from 12 days (Figure 3F) to potentially 3 weeks, allowing for long-term studies of neuronal-microglia interactions45.
While the relative simplicity of the co-culture system is advantageous for accessibility, a key limitation of this approach is that the two-dimensional cell culture does not fully represent the CNS tissue environment. Indeed, a tri-culture of microglia, neurons, and astrocytes is shown to mimic in vivo neuroinflammatory response more reliably than microglia or astrocyte-neuronal co-culture46,47. Furthermore, recent developments in brain organoid systems with microglia and other glial cells, including astrocytes and oligodendrocytes, are more suitable for studying three-dimensional (3D) tissue architecture and interactions of the CNS48. Similarly, a human 3D triculture system comprising neurons, microglia, and astrocytes is shown to be effective in modeling Alzheimer&#39;s disease condition49. Other human microglia-neuron co-cultures may be more physiologically relevant to neurodegenerative disease studies. For example, the co-culture of microglia and motor neurons derived from human induced pluripotent stem cells50 may be useful for studying diseases such as amyotrophic lateral sclerosis51. Nevertheless, given the relative abundance and low cost of generating primary neurons and microglia from mice, assessing microglia-neuronal co-culture in 96-well plates can be a high throughput approach to studying the mechanisms and functional implications of microglia-neuronal interactions. Downstream assays may include using live cell imaging to determine cellular functional changes in real-time, RNA sequencing to determine cellular transcriptional changes, immunofluorescence microscopy to determine cellular phenotypic changes, mass spectrometry to determine cellular&#160;and proteomic changes, or enzyme-linked immunosorbent assay and western blot to measure protein secreted into the co-culture supernatant.
As microglia are important mediators of CNS health and diseases, this co-culture can also be used to investigate the effect of disease-related molecules and compounds on microglia-neuronal bi-directional communication. In the context of MS, activated microglia may take on a destructive role, contributing to the worsening of pathology52,53. For example, they may secrete pro-inflammatory cytokines and reactive oxygen species, as well as promote astrocyte activation45. This protocol may help to further interrogate the harmful role of microglia by treating microglia in the co-culture with inflammatory cytokines found elevated in MS and assessing the outcomes of neuron health. Alternatively, this microglia-neuronal co-culture has been used to show the protective effect of microglia on neurons in response to oxidized phosphatidylcholine, an oxidative product discovered in MS54. Moreover, neuronal apoptosis remodels microglia gene expression during development by promoting microglia survival and phagocytosis55. Therefore, this protocol may also help to determine the impact of neuronal death on microglia phenotype and function.
In summary, this protocol describes the generation and co-culturing of primary mouse-derived neurons and microglia in vitro. The microglia-neuronal co-culture set up in 96-well plates can be a high throughput approach to interrogate the mechanisms and function of microglia interactions with neurons in the context of CNS health and disease, including MS.

Microglia are tissue-resident macrophages that facilitate immunosurveillance and homeostasis in the central nervous system (CNS)1,2,3. They originate from yolk sac erythromyeloid progenitor cells that colonize the brain during embryonic development4,5,6&#160;and are maintained throughout the organism&#39;s life span through self-renewal, which involves proliferation and apoptosis7. At steady-state, resting microglia have ramified morphology and engage in tissue surveillance8,9,10.
Microglia express numerous cell-surface receptors, which enables them to rapidly respond to changes in the CNS11,12 and to promote inflammatory responses in the event of infections or tissue injury12,13,14, as well as during neurodegenerative diseases9,15, such as&#160;multiple sclerosis (MS)16,17. Microglia also express receptors to various neurotransmitters and neuropeptides18,19,20, which suggests they may also respond to and regulate neuronal activity21,22. Indeed, microglia and neurons interact in various forms of bidirectional communication8,23 such as direct interactions mediated by membrane proteins or indirect interactions through soluble factors or intermediate cells23,24.
For instance, various neurotransmitters secreted by neurons can modulate the neuroprotective or inflammatory activity of microglia25,26,27. Additionally, direct interactions between neurons and microglia help to maintain microglia in a homeostatic state28. Conversely, direct interactions of microglia with neurons can shape neuronal circuitry29 and influence neuronal signaling30,31,32. As disruptions of these interactions induce hyperexcitability of neurons30 and&#160;microglia reactivity33,34, dysregulated microglia-neuronal interactions&#160;are implicated&#160;as a contributing factor to&#160;neurological diseases33,35. Indeed, psychotic23,26 and neurodegenerative diseases have been described to exhibit dysfunctional microglia-neuronal interactions33. While these observations highlight the importance of microglia-neuronal communication in the CNS, specific mechanisms of how these interactions regulate microglial and neuronal functions in health and disease are relatively unknown.
Within a complex milieu such as the CNS, multiple environmental factors can influence microglia-neuronal interactions, which limits the ability to study transient cellular interactions in vivo. Here, we present an in vitro microglia-neuronal co-culture system that can be used to study direct cellular interactions between microglia and neurons. This protocol describes the generation of primary microglia and neurons from the cortices of neonatal mice between post-natal days 0-2 and embryonic mice days 16-18, respectively. Neurons and microglia are then co-cultured in 96-well plates for downstream high-throughput experiments. We previously used this approach to demonstrate that microglia phagocytosis protects neurons from oxidized phosphatidylcholine mediated cell death37, suggesting that this method can help to understand the roles of microglia in the context of neurodegeneration and MS. Similarly, microglia-neuronal co-cultures may also be useful for investigating the impact of microglia-neuronal crosstalk in other contexts such as viral infections38 or neuronal injury and repair39. Overall, in vitro microglia-neuronal co-culture systems&#160;enable researchers to study microglia-neuronal interactions in a manipulatable and controlled environment, which complements in vivo models.

<table><tbody><tr><td>10 cm Petri dish&amp;nbsp;</td><td>Fisher&amp;nbsp;</td><td>07-202-011</td><td>Sterile</td></tr><tr><td>1x Versene</td><td>Gibco</td><td>15040-066</td><td></td></tr><tr><td>B-27 Plus Neuronal Culture System&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>A3653401</td><td></td></tr><tr><td>Dissection microscope</td><td>VWR</td><td></td><td></td></tr><tr><td>DNase I</td><td>Roche</td><td>11284932001</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>11960-044</td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>ThermoFisher Sci</td><td>12483-020</td><td></td></tr><tr><td>HBSS (10x)</td><td>Gibco</td><td>14065-056</td><td></td></tr><tr><td>Hemacytometer</td><td>Hausser Scientific</td><td>1475</td><td></td></tr><tr><td>HEPES&amp;nbsp;</td><td>ThermoFisher Sci</td><td>15630080</td><td></td></tr><tr><td>Leibovitz&amp;rsquo;s L-15 Medium (1x)</td><td>Fisher Scientific&amp;nbsp;</td><td>21083027</td><td></td></tr><tr><td>Macrophage colony stimulating factor&amp;nbsp;</td><td>Peprotech</td><td>315-02</td><td></td></tr><tr><td>Micro-Forceps</td><td>RWD</td><td>F11020-11</td><td>Autoclaved/Sterile</td></tr><tr><td>Non-essential amino acids</td><td>Cytiva</td><td>SH3023801</td><td></td></tr><tr><td>PBS (10x)</td><td>ThermoFisher Sci</td><td>AM9625</td><td></td></tr><tr><td>Penicillin Streptomycin Glutamine (100x)</td><td>Gibco</td><td>103780-16</td><td></td></tr><tr><td>Poly-L-ornithine hydrobromide&amp;nbsp;</td><td>Sigma</td><td>P3655-100MG</td><td></td></tr><tr><td>Sodium pyruvate (100 mM)</td><td>Gibco</td><td>11360-070</td><td></td></tr><tr><td>Spring scissors</td><td>RWD</td><td>S11008-42</td><td>Autoclaved/Sterile</td></tr><tr><td>Surgical blade</td><td>Feather</td><td>08-916-5D</td><td>Sterile</td></tr><tr><td>T-25 flasks</td><td>Fisher</td><td>10-126-9</td><td></td></tr><tr><td>T-75 flasks&amp;nbsp;</td><td>Fisher</td><td>13-680-65</td><td></td></tr><tr><td>Tissue forceps</td><td>Codman</td><td>30-4218</td><td>Autoclaved/Sterile</td></tr><tr><td>Tissue scissors</td><td>RWD</td><td>S12052-10</td><td>Autoclaved/Sterile</td></tr><tr><td>Trypan Blue&amp;nbsp;</td><td>Thermofisher Sci&amp;nbsp;</td><td>15250-061</td><td></td></tr><tr><td>Trypsin (2.5%)</td><td>ThermoFisher Sci</td><td>15090046</td><td></td></tr><tr><td>Widefield Immunofluorescence Microscope</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

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.

A flowchart showing the key steps of the mixed glia culture for microglia is shown in Figure 1A. Overall, sparse cells and excessive cellular debris are expected on day 1 (Figure 1B). By day 4, increased cell number should be observed, especially with the generation of adherent astrocytes, as indicated by their elongated morphology (Figure 1C). A few microglia may be observed on top of the astrocytes or as small round cells floating in media. By day 8, a confluent layer of astrocytes will form the adherent layers of cells, whereas microglia will appear as round and bright cells that are semi-attached to the astrocytes or floating in media (Figure 1D). If microglial density is low by day 8, the culture may be incubated for additional days before harvest.
A flow chart showing the key steps of the neuronal culture is shown in Figure 2A. In a healthy culture, neurons will attach and form neurite processes after 1-3 days in vitro (Figure 2B). Similarly, upon seeding neurons into a 96-well plate, they will appear round initially, but by day 3, neurons should become adherent and form neurite processes that connect with neighboring neurons (Figure 2C). Conversely, unhealthy neuron cultures will contain cell clumps and cell debris, which can result from deficient cell dissociation or mishandling of neurons (Figure 2D).
A flow chart showing the key steps of the microglia-neuronal co-culture protocol is shown in Figure 3A. After adding 2.5 x 104 microglia to 7.5 x 104 neurons, round and large microglia should appear on top or between the neurons and neurite processes (Figure 3B). To further visualize the microglia-neuronal co-culture, microglia and neurons alone or in co-culture were labeled with ionized calcium-binding adaptor molecule 1 (IBA-1, red) and tubulin βIII (TUJ1, green) antibodies37 for immunofluorescence microscopy (Figure 3C-F).
<img alt="Neuronal cell culture process diagram; microscopy images show cell suspension, flasks, incubator results." class="xfigimg" src="/files/ftp_upload/67078/67078fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Overview of primary microglia culture. (A) Flow chart of the key steps in generating a mixed glia culture. (B-D) Representative 10x bright-field microscope images of mixed glia culture on day 1 (B), day 4 (C), and day 8 (D). Scale bars: 100 µm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67078/67078fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Neuronal culture process in T25 flasks and 96-well plate; embryonic mouse cells; microscopy." class="xfigimg" src="/files/ftp_upload/67078/67078fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Overview of primary neuron culture. (A) Flow chart of the key steps in generating neurons. (B) Representative 10x bright field microscope images showing neurons cultured in T-25 flasks. (C) Representative 10x bright field microscope of neurons cultured in a 96-well plate. (D) Representative 10x bright field microscope of an unhealthy neuronal cluster in a 96-well plate. Scale bars: 100 µm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67078/67078fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microglia assay workflow, microscopy images, immunofluorescence, cell staining, neuronal culture." class="xfigimg" src="/files/ftp_upload/67078/67078fig03.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Overview of microglia-neuron co-culture. (A) Flow chart of the key steps for the microglia-neuronal co-culture. (B) Representative 10x bright-field microscope images of microglia-neuronal co-culture in 96-well plates. Scale bars: 100 µm. (C,D) Representative immunofluorescence microscopy images of neurons alone (C), microglia alone (D), day 2 neuron and microglia co-culture (E), or 12-day neuron and microglia co-culture (F) in a 96-well plate. Microglia and neurons are labeled with IBA-1 (red) and tubulin βIII (green), respectively. Nuclei are stained with DAPI (blue). Immunofluorescence images were acquired using a 25x, 0.8 NA, water immersion objective with a widefield fluorescence microscope. Scale bars: 50 µm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67078/67078fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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.

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 ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

1.9K Views.  University of Saskatchewan. Ons onderzoek is gericht op het onderzoeken van de interacties tussen neuronen en microglia tijdens ziektetoestanden zoals multiple sclerose. We streven ernaar om microglia neuronale interacties in vitro te recapituleren om hun functionele belang in het CZS beter te begrijpen. We hebben eerder aangetoond dat microglia-fagocytose neuronen beschermt tegen geoxideerde fosfatidylcholine-gemedieerde celdood met deze co-cultuur.Hoewel ook andere co-cultuursy...

Video: Auteur in de schijnwerpers: In vitro co-cultuurmodel voor het bestuderen van microglia-neuronale interacties bij ziektetoestanden

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

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

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

Primary Microglia Isolation from Postnatal Mouse Brains

Microglia are the mononuclear phagocytes in the central nervous system (CNS), which play key roles in maintaining homeostasis and regulating the inflammatory process in the CNS. To study the microglial biology in vitro, primary microglia show great advantages compared to immortalized microglial cell lines. However, microglia isolation from the postnatal mouse brain is relatively less efficient and time-consuming. In this protocol, we provide a quick and easy-to-follow method to isolate primary microglia from the neonatal mouse brain. The overall steps of this protocol include brain dissection, primary brain cell culture, and microglia isolation. Using this approach, researchers can obtain primary microglia with high purity. In addition, the harvested primary microglia were able to respond to the lipopolysaccharides challenge, indicating they retained their immune function. Collectively, we developed a simplified approach to efficiently isolate primary microglia with high purity, which facilitates a wide range of microglial biology investigations&#160;in vitro.

Primary cell culture is one of the primarily used approaches for studying microglial biology in vitro. Here, we developed a method for simple and rapid microglia isolation from the mouse postnatal day 1 (P1) to P4.

Microglia are the mononuclear phagocytes in the central nervous system (CNS), which play key roles in maintaining homeostasis and regulating the ...

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 ...

Embryonic Mouse Brain Mixed Cell Cultures to Study Infection and Innate Immunity

Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of neurons, astrocytes, oligodendrocytes, and microglia. Due to increasing efforts to replace and reduce animal use, a variety of in vitro cell culture systems have been developed to explore innate cell properties, which allow the development of therapeutics for CNS infections and pathologies. Whilst certain research questions can be addressed by human-based cell culture systems, such as (induced) pluripotent stem cells, working with human cells has its own limitations with regard to availability, costs, and ethics. Here, we describe a unique protocol for isolating and culturing cells from embryonic mouse brains. The resulting mixed neural cell cultures mimic several cell populations and interactions found in the brain in vivo. Compared to current equivalent methods, this protocol more closely mimics the characteristics of the brain and also garners more cells, thus allowing for more experimental conditions to be investigated from one pregnant mouse. Further, the protocol is relatively easy and highly reproducible. These cultures have been optimized for use at various scales, including 96-well based high throughput screens, 24-well microscopy analysis, and 6-well cultures for flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. This culture method is a powerful tool to investigate infection and immunity within the context of some of the complexity of the CNS with the convenience of in vitro methods.

Author Spotlight: Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity  

This protocol presents a unique way of generating central nervous system cell cultures from embryonic day 17 mouse brains for neuro(immuno)logy research. This model can be analyzed using various experimental techniques, including RT-qPCR, microscopy, ELISA, and flow cytometry.

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of ...

Coculturing AdMSCs and Glia to Reduce Prion-Induced Inflammation

Adipose-Derived Mesenchymal Stromal Cells Co-Cultured with Primary Mixed Glia to Reduce Prion-Induced Inflammation

Mesenchymal stromal cells (MSCs) are potent regulators of inflammation through the production of anti-inflammatory cytokines, chemokines, and growth factors. These cells show an ability to regulate neuroinflammation in the context of neurodegenerative diseases such as prion disease and other protein misfolding disorders. Prion diseases can be sporadic, acquired, or genetic; they can result from the misfolding and aggregation of the prion protein in the brain. These diseases are invariably fatal, with no available treatments.
One of the earliest signs of disease is the activation of astrocytes and microglia and associated inflammation, which occurs prior to detectable prion aggregation and neuronal loss; thus, the anti-inflammatory and regulatory properties of MSCs can be harvested to treat astrogliosis in prion disease. Recently, we showed that adipose-derived MSCs (AdMSCs) co-cultured with BV2 cells or primary mixed glia reduce prion-induced inflammation through paracrine signaling. This paper describes a reliable treatment using stimulated AdMSCs to decrease prion-induced inflammation.
A heterozygous population of AdMSCs can easily be isolated from murine adipose tissue and expanded in culture. Stimulating these cells with inflammatory cytokines enhances their ability to both migrate toward prion-infected brain homogenate and produce anti-inflammatory modulators in response. Together, these techniques can be used to investigate the therapeutic potential of MSCs on prion infection and can be adapted for other protein misfolding and neuroinflammatory diseases.

Author Spotlight: Insight Into Advances in Prion Diseases Research

Adipose-derived mesenchymal stromal cells (AdMSCs) have potent immunomodulatory properties useful for treating diseases associated with inflammation. We demonstrate how to isolate and culture murine AdMSCs and primary mixed glia, stimulate AdMSCs to upregulate anti-inflammatory genes and growth factors, assess migration of AdMSCs, and co-culture AdMSCs with primary mixed prion-infected glia.

Mesenchymal stromal cells (MSCs) are potent regulators of inflammation through the production of anti-inflammatory cytokines, chemokines, and growth ...

Generating a Primary Culture of Astrocytes from a Rat Pup Brain

Source: Mili&#263;evi&#263;, K., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63483">Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis.</a> J. Vis. Exp. (2022)
The video demonstrates a technique for culturing primary astrocytes from the rat cortex. The cortex is mechanically dissociated to obtain a homogeneous glial cell suspension, which is then plated on polypeptide-coated plates to promote selective proliferation of astrocytes. Subsequently, the astrocytes are enzymatically dissociated from the plates and transferred to fresh media, allowing the culture of pure astrocytes.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Primary cell culture preparation

...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

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 ...

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

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