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 c...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 cm Petri dish&#160;</td>
			<td>Fisher&#160;</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&#160;</td>
			<td>Gibco&#160;</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&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11960-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;</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&#160;</td>
			<td>ThermoFisher Sci</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#8217;s L-15 Medium (1x)</td>
			<td>Fisher Scientific&#160;</td>
			<td>21083027</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrophage colony stimulating factor&#160;</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&#160;</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&#160;</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&#160;</td>
			<td>Thermofisher Sci&#160;</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.

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

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Our research aims to investigate the interactions between neurons and microglia during disease conditions such as multiple sclerosis. We aim to recapitulate microglia neuronal interactions in vitro to better understand their functional importance in the CNS. We previously demonstrated that microglia phagocytosis protects neurons from oxidized phosphatidylcholine-mediated cell death with this co-culture.While other co-culture systems are also employed, this mixed culture system enables direct and close contact between microglia and neurons. In addition, it can remain healthy from 12 days to potentially three weeks, allowing for long-term studies of neuronal microglia interactions. Our protocol offers a simple and accessible method to co-culture microglia neurons that are easily manipulatable and can be further used in downstream assays to determine cellular functional changes.

A flowchart showing the key steps of the mixed glia&#160;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 flo...

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 ...

generating-co-culturing-murine-primary-microglia-cortical

Research

JoVE Journal

Neuroscience

Brain Isolation from Neonatal Mice for Primary Microglia Culture

To begin, add 5 milliliters of 10 micrograms per milliliter Poly-L-ornithine hydrobromide to each T75 flask. Incubate at 37 degrees Celsius in a 5%carbon dioxide incubator for at least one hour. After decapitating the mouse, gently hold the head and 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. To scoop the brain out, insert the tissue forceps under the brain, and lift the brain out from the head. After placing the brain into a Petri dish containing 20 milliliters of Leibovitz's L-15 media under a dissection microscope, using a pair of micro-forceps, carefully remove the brainstem and the meninges.Collect the brains in a 50-milliliter conical tube containing 5 milliliters of Leibovitz's L15 media.

Preparing and Maintaining Mixed Glia Culture Derived from Neonatal Mice Brains

To begin, mince the isolated brains into approximately one-square-millimeter pieces with a sterile disposable scalpel blade. Carefully transfer the minced brains into a new sterile 50 milliliter tube. Add a final concentration of 0.25%trypsin to the minced brains.Incubate the tissue in a 37-degrees-Celsius water bath for 20 minutes. Add five milliliters of complete glia culture media to neutralize the trypsin and gently mix the tissue suspension using a 10 milliliter pipette. Carefully decant the cell suspension through the cell strainer.Then remove the plunger from a sterile one-milliliter syringe and grind the tissue into the mesh using the plunger. Add equal volumes of cell suspension containing one to two brains with glia culture media to each flask resulting in a final volume of 15 milliliters. Sparse cells and excessive cellular debris were observed on day one.By day four, adherent astrocytes were generated. Confluent cells with typical astrocyte morphology were observed by day eight of culture.

Isolating Brains from E16-E18 Mouse Embryos for Primary Neuron Culture

Begin by coating T25 flasks with five milliliters of 10 micrograms per milliliter poly-L-ornithine, and incubate them. To isolate the brain from mouse embryos, lift the skin of the euthanized pregnant mouse's lower abdomen with tissue forceps. Using dissection scissors, perform a V-shaped incision from this point to the lower rib cage.Grab the uterine horns containing the embryos with tissue forceps, and gently remove the embryos from the abdominal cavity. Using spring scissors, create a midline incision at the top of the skull, starting from the back of the neck to the nose. 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 centimeter Petri dish containing 15 milliliters of Hanks'balanced salt solution. Under a dissection microscope, carefully remove the brainstem and the meninges on both sides of the brain cortices with a pair of micro forceps.

Brain Dissociation and Seeding of Cortical Neurons in Cell Culture 

To begin, mince the isolated brains into pieces in the 50-milliliter conical tube with a sterile disposable scalpel blade. Add trypsin to the tube at a final concentration of 0.25%Immerse the tube in a 35 to 38 degrees Celsius hot water bath for 15 minutes. Gently pipette the tissue suspension up and down to dissociate cells.Then add 0.5 milliliters of fetal bovine serum to the homogenized suspension to neutralize trypsin activity. Filter the cell suspension through the 70-micrometer nylon mesh cell strainer. Using a one-milliliter syringe plunger, gently grind the tissue into the cell strainer.Then centrifuge the conical tube at 300 g for five minutes at four degrees Celsius. Discard the Poly-L-Ornithine in the T25 flasks and rinse the flasks three times with five milliliters of PBS. Carefully decant the tube to remove the supernatant and resuspend the cell pellet by gently pipetting with two to three milliliters of complete neurobasal media.Plate the cells in the Poly-L-Ornithine-coated T25 flasks with approximately 20 million cells per flask in five milliliters of complete neurobasal media. Incubate the cell culture at 37 degrees Celsius in a 5%carbon dioxide incubator. Neurons in T25 flasks and 96-well plates adhered and formed neurite processes by day three.Suboptimal neuron cultures contained cell clumps and debris.

Co-Culture of Primary Neurons and Microglia for Studying Microglia-Neuron Interactions

To begin, take neurons grown in T25 flasks for co-culture. Replace the media with five milliliters of versene solution containing trypsin and Dnase I.Add 0.5 milliliters of fetal bovine serum to neutralize the trypsin and Dnase I.Transfer cells to a 15 milliliter conical tube. Wash the flask with five milliliters of complete neurobasal media once to maximize the collected neurons.Then, centrifuge the cell suspension at 300 G for five minutes at four degrees celsius. After discarding the supernatant, gently resuspend the cell pellet in two to three milliliters of complete neurobasal media. Seed 100 microliters of the cell suspension to each well to obtain 75, 000 neurons per well, between eight to 10 days of mixed glia cultivation.Using a 10 milliliter pipette, gently wash the T75 flasks with glia culture media to collect microglia and transfer the cells and media into a sterile 50 milliliter conical tube. Centrifuge the cell collection at 300 G for five minutes at four degrees celsius. Once the supernatant is removed, gently resuspend the pellet in two milliliters of complete neurobasal media.From the 96-well neuronal culture plate, remove 100 microliters of neurobasal media and add 100 microliters of 250, 000 cells per milliliter cell suspension. In co-culture round and large microglia were observed on top or between neurons and neurite processes.

626 Views.  University of Saskatchewan.  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.

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

Summary