We would like to thank members of the Edgar and Linington labs, particularly Prof. Chris Linington, Dr&#160;Diana Arseni, and Dr&#160;Katja Muecklisch, for their advice, helpful comments, and assistance with feeding the cultures while we set up these cultures. Particular thanks go to Dr Muecklisch for providing the starting points for the Cell Profiler pipelines. This work was supported by the MS Society (grant 122) and the Yuri and Lorna Chernajovsky foundation to MP; University of Glasgow funding to JC and MP; and Wellcome Trust (217093/Z/19/Z) and Medical Research Council (MRV0109721) to GJG.

All animal experiments complied with local laws and guidelines for animal use, and were approved by the local Ethical Review Committee at the University of Glasgow. Animals were housed in specific pathogen-free conditions in accordance with the UK Animals Scientific Procedures Act 1986, under the auspices of a UK Home Office Project License. For this study, in-house bred adult C57BL/6J mice were used. The use of young females (8-12 weeks) is recommended due to the higher success rate of pregnancy; males can be reused for multiple rounds of breeding. Figure 1 represents a schematic overview of the described method to generate mixed neuronal and glial cultures.
1. Preparation of the tissue culture consumables
<ol>
	<li>Prepare the plates and/or dishes containing microscopy coverslips inside a Class 2 Safety cabinet. Sterilize all reagents or autoclave to ensure sterility during the culture period.</li>
	<li>Add the appropriate volume of BA-PLL (13.2 &#181;g/mL poly-L-lysinehydrobromide [PLL] in boric acid buffer [BA] [50 mM boric acid, 12.5 mM sodium tetraborate, pH 8.5]; see Table of Materials) to each well (1,000 &#181;L/well in a 6-well plate, 100 &#181;L/well in a 96-well plate; volumes are summarized in Table 1). For microscopy coverslips, add 20 mL of BA-PLL to a 9 cm diameter tissue culture dish containing 200 sterile coverslips, and swirl to distribute evenly.</li>
	<li>Incubate at 37 &#176;C for 1-2 h.</li>
	<li>Remove the BA-PLL solution from each well or dish containing microscopy coverslips, and wash by adding 20 mL of sterile water, swirling the coverslips, and then removing the water. Repeat this wash step three times. For a dish containing coverslips, leave sterile water in the tissue culture dish on the final wash to easily remove the coverslips.</li>
	<li>Remove as much liquid as possible with a sterile pipette and allow to dry for at least 2 h to&#160;overnight.</li>
	<li>Store the coated plates at 4 &#176;C for up to 2 months. 
	NOTE: The prepared boric acid with poly-l-lysine (BA-PLL) solution can be reused up to three time, adding new PLL each time. Store the BA-PLL at 4 &#176;C. Dishes are treated with BA-PLL, as the PLL allows the cells to stick down and grow. Without this treatment, the cells will lift after approximately 1 week of culture and no longer be able to differentiate.</li>
</ol>
2. Dissection of E17 embryonal brains
<ol>
	<li>Co-house one or multiple female mice with a male mouse. Check the females daily for a mucus plug, indicating mating has taken place. 
	NOTE: Any &#34;plugged&#34; female mice need to be separated from the male to ensure the correct start date of gestation. Mice can be weighed to confirm pregnancy or monitored visually.</li>
	<li>Cull the pregnant mouse at E17 using appropriate methods in compliance with local animal welfare guidance and laws, for example, by raising carbon dioxide (CO2) concentration, a lethal anesthetic overdose, or dislocation of the neck. 
	NOTE: The chosen method must not disrupt the embryos. For this study, exposure to a rising concentration of carbon dioxide gas followed by confirmation of death by severing the femoral artery was used to cull pregnant dams.</li>
	<li>Place the culled, pregnant mouse on its back on a dissection board; while pinning it down is not required, it might make it easier for inexperienced researchers. Pinch the midline of the abdomen using forceps. Using sharp scissors, cut open the abdomen through the skin and the peritoneum over the midline from the genitalia to the ribcage, being careful not to puncture the uterus.
	<ol>
		<li>The mouse uterus has two horns, each typically containing one to five embryos. Remove the uterus containing the embryos from the mother and immediately place it on ice.</li>
	</ol>
	</li>
	<li>Cut through the yolk sack on the side of the placentas, being careful not to damage the embryos, and remove the embryos from their yolk sack.</li>
	<li>Immediately decapitate the embryos. Add the decapitated heads into a dish with Hanks&#39; balanced salt solution (HBSS) without calcium (Ca2+) and magnesium (Mg2+) (HBSS-/-) on ice. 
	NOTE: If genotyping is required, one can remove the tail at this stage for genetic analysis. When multiple genotypes are expected, the heads of each embryo should be kept separately for culturing.</li>
	<li>Using angled forceps, position the head on its side facing left.</li>
	<li>Pierce the eye with one edge of the forceps, firmly holding the chin with the other.</li>
	<li>Starting at the nape, gently tear the skin of the scalp along the midline toward the tip of the snout.</li>
	<li>Entering through the spinal cord, noticeable as a white oval, use the angled forceps to crack open the skull along the midline, exposing the brain.</li>
	<li>Gently peel the skull away on the side facing upward, exposing the brain.</li>
	<li>Lift the brain out of the skull, disposing of the skull once the brain has been completely removed.</li>
	<li>Using the forceps, remove the meninges, which are noticeable as a thin membrane with dense blood vessels.</li>
	<li>Place the brains into a bijou (see Table of Materials) containing 2 mL of HBSS-/- on ice.</li>
	<li>Repeat steps 2.6 to 2.13 with the remaining brains, adding up to four brains per bijou.</li>
	<li>Add 250 &#181;L of 10x trypsin to the bijou and triturate the brains by shaking the bijou. Incubate for 15 min at 37 &#176;C. 
	NOTE: All steps from this point forward should be performed in a sterile tissue culture hood.</li>
	<li>Thaw 2 mL of soybean trypsin (SD) inhibitor (Leibovitz L-15, 0.52 mg/mL trypsin inhibitor from soybean, 40 &#181;g/mL DNase I, 3 mg/mL bovine serum albumin [BSA] fraction V; see Table of Materials) from -20 &#176;C by placing it at 37 &#176;C.</li>
	<li>Add 2 mL of SD inhibitor to each bijou containing brains (per bijou containing up to four brains), shaking the bijou again to disperse it evenly. 
	NOTE: SD inhibitor decreases the trypsin activity to prevent unnecessary digestion of the samples and preserve cell viability.</li>
	<li>Without centrifugation, remove 2 mL of the supernatant from each bijou and transfer into a 15 mL centrifuge tube, being careful not to transfer cell clumps.</li>
	<li>Triturate the remaining cells in the bijou with a 19 G needle attached to a 5 mL syringe by aspirating the suspension twice. This will create a thick mucus-like mixture.
	<ol>
		<li>Repeat twice more using a 21 G needle. If there are clumps remaining, triturate once more with the 21 G needle.</li>
	</ol>
	</li>
	<li>Transfer the cells from the bijou to the same 15 mL centrifuge tube (from step 2.18) using a 23 G needle.</li>
	<li>Centrifuge at 200 x g at room temperature (RT) for 5 min.</li>
	<li>Remove all the supernatant using a 5 mL serological pipette and transfer it to another 15 mL centrifuge tube, being careful not to disturb the loose pellet at the bottom containing the required cells.</li>
	<li>Centrifuge the supernatant again at 200 x g at RT for 5 min. 
	NOTE: This step is not essential, but if one requires many cells or has few embryos, one could perform this step to recover as many cells as possible from the supernatant.</li>
	<li>Using 10 mL of plating media (PM) (49% Dulbecco&#39;s modified eagle medium [DMEM], 1% penicillin/streptomycin [Pen/Strep], 25% horse serum, 25% HBSS with Ca2+ and Mg2+ [HBSS+/+]; see Table of Materials), combine and resuspend the two pellets together to create a whole cell suspension.</li>
	<li>Count the cells using trypan blue and either a hemocytometer or digital cell counter, and dilute the cell suspension with PM to a concentration of 1.8 x 106 cells/mL.</li>
</ol>
3. Plating the cells
<ol>
	<li>Add the required volume of the cell suspension to the required format as detailed in Table 1: 1,000 &#181;L per well in 6-well format, 50 &#181;L per well in 96-well format, or 100 &#181;L per coverslip.</li>
	<li>Incubate for 2-4 h at 37 &#176;C with 5%-7% CO2. Check the cells have adhered using an inverted microscope.</li>
	<li>Remove the media and top up with new differentiation media (DM+: DM- including 10 &#181;g/mL insulin. DM-: DMEM, 1% Pen/Strep, 50 nM hydrocortisone, 10 ng/mL biotin, 2.5 mL 100x N1 media supplement; see Table of Materials). Volumes are detailed in Table 1. Press down any floating coverslips using a sterile pipette tip.</li>
</ol>
4. Maintaining the cultures
NOTE: These cultures require feeding thrice weekly to support optimal growth and differentiation. The cultures will reach optimum health and maturity for experiments on DIV (days in vitro) 21. Cells can be kept in culture for up to 28 days, after which the cultures quickly degenerate.
<ol>
	<li>Three times per week until DIV12, replace part of the supernatant with fresh DM+ by removing 500 &#181;L per well in 6-well format, 50 &#181;L per well in 96-well format, or 500 &#181;L per dish containing three coverslips, and adding 600 &#181;L per well in 6-well format, 60 &#181;L per well in 96-well format, or 600 &#181;L per coverslip dish (Table 1).</li>
	<li>Three times per week from DIV13 onwards, replace part of the supernatant with fresh DM- by removing 500 &#181;L per well in 6-well format, 50 &#181;L per well in 96-well format, or 500 &#181;L per dish containing three coverslips, and adding 600 &#181;L per well in 6-well format, 60 &#181;L per well in 96-well format, or 600 &#181;L per coverslip dish (Table 1).</li>
</ol>

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

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E., 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=synapsing+cultures+of+murine+spinal+cord-validation+as+an+in+vitro+model+of+the+central+nervous+system.">synapsing cultures of murine spinal cord-validation as an in vitro model of the central nervous system.</a> The European Journal of Neuroscience. 28 (8), 1518-1535 (2008).</li><li>Bijland, S., 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=An+in+vitro+model+for+studying+CNS+white+matter:+Functional+properties+and+experimental+approaches.">An in vitro model for studying CNS white matter: Functional properties and experimental approaches.</a> F1000Research. 8, 117 (2019).</li><li>Fragkoudis, R., 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=Neurons+and+oligodendrocytes+in+the+mouse+brain+differ+in+their+ability+to+replicate+Semliki+Forest+virus.">Neurons and oligodendrocytes in the mouse brain differ in their ability to replicate Semliki Forest virus.</a> Journal of Neurovirology. 15 (1), 57-70 (2009).</li><li>Hayden, L., 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=Lipid-specific+IgMs+induce+antiviral+responses+in+the+CNS:+Implications+for+progressive+multifocal+leukoencephalopathy+in+multiple+sclerosis.">Lipid-specific IgMs induce antiviral responses in the CNS: Implications for progressive multifocal leukoencephalopathy in multiple sclerosis.</a> Acta Neuropathologica Communications. 8 (1), 135 (2020).</li><li>Kantzer, C. 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The authors have nothing to disclose.

The CNS is a complex network that spans from the brain down to the spinal cord and consists of many cell types, predominantly neurons, oligodendrocytes, astrocytes, and microglia1. As each cell has an important role in maintaining homeostasis and generating appropriate responses to challenges in the CNS9,10,11, a culture system that contains all these cell types is a useful and versatile tool to investiga...

Improving our understanding of the central nervous system (CNS) is critical to improve therapeutic options for many neuroinflammatory and neurodegenerative diseases. The CNS, a complex network of interconnected cells within the brain, spinal cord, and optic nerves, comprises neurons, oligodendrocytes, astrocytes, and their innate immune cells, the microglia1. An in vitro approach can often drastically reduce the number of mice required to perform meaningful research; however, the complex nature of the CNS makes it impossible to recapitulate the in vivo situation using cell lines. Mixed neural cell cultures provide an extremely valuable research tool to investigate neuro(immuno)logy questions in a relevant model, in line with the Replacement, Reduction and Refinement (3Rs) principles2,3.
Thomson et al. described a cell culture method using prenatal spinal cord cells that differentiate into all the aforementioned main CNS cell types4. This system also has synapse formation, myelinated axons, and nodes of Ranvier. The main limitation of this culturing method is that, being spinal cord, it does not usefully model the brain, and the cell yields from embryonic day 13 (E13) spinal cords are constricting. Thus, this limits the number of experimental conditions that can be investigated. Therefore, this study aimed to develop a new cell culture system that recapitulates the characteristics of the brain with increased cell yield to reduce the requirements for animals.
Using Thomson et al. as a starting point, we developed a cell culture model derived purely from prenatal mouse brains. These cultures have the same cell populations, interconnectivity, and treatment options as the spinal cord cultures, except there is less myelination by comparison. However, having a CNS in vitro model with an approximately threefold higher cell yield is more efficient, requiring fewer mice and less time processing embryos. We optimized this unique culture system for multiple downstream applications and scales, including using glass coverslips for microscopy analysis and various sizes of plastic well plates, including 96-well plates for high throughput research.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x Trypsin</td>
			<td>Sigma</td>
			<td>T4549-100ML</td>
			<td>To digest tissue</td>
		</tr>
		<tr>
			<td>140 mm TC Dish</td>
			<td>Fisher</td>
			<td>11339283</td>
			<td>Put 8 35 mm dishes per 1 140 mm dish</td>
		</tr>
		<tr>
			<td>15 mL Falcon</td>
			<td>Sarstedt</td>
			<td>62554502</td>
			<td>To collect cells into pellet for resuspension in plating media</td>
		</tr>
		<tr>
			<td>18 G needle</td>
			<td>Henke Sass Wolf</td>
			<td>4710012040</td>
			<td>For trituration of sample</td>
		</tr>
		<tr>
			<td>21 G needle</td>
			<td>BD</td>
			<td>304432</td>
			<td>For trituration of sample</td>
		</tr>
		<tr>
			<td>23 G needle</td>
			<td>Henke Sass Wolf</td>
			<td>4710006030</td>
			<td>For trituration of sample</td>
		</tr>
		<tr>
			<td>35 mm TC Dish</td>
			<td>Corning</td>
			<td>430165</td>
			<td>Plate out 3 PLL coated coverslips per 1 35 mm dish</td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>Fisher</td>
			<td>15869152</td>
			<td>For trituration of sample</td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Corning</td>
			<td>3516</td>
			<td>To plate out cells for RT-qPCR, and flow cytometry</td>
		</tr>
		<tr>
			<td>7 mL Bijoux</td>
			<td>Fisher</td>
			<td>DIS080010R</td>
			<td>To put brains intp</td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>Corning</td>
			<td>3596</td>
			<td>To plate out cells for high-throughput testing</td>
		</tr>
		<tr>
			<td>ACSA-2 Antibody, anti-mouse, PE</td>
			<td>Miltenyi</td>
			<td>130-123-284</td>
			<td>For flow cytometry staining of astrocytes</td>
		</tr>
		<tr>
			<td>Angled forceps</td>
			<td>Dumont</td>
			<td>0108-5/45-PO</td>
			<td>For dissection</td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Sigma</td>
			<td>B4501</td>
			<td>For DM+/-</td>
		</tr>
		<tr>
			<td>Boric Acid</td>
			<td>Sigma</td>
			<td>B6768-500G</td>
			<td>For boric acid buffer</td>
		</tr>
		<tr>
			<td>Brilliant Violet 421 anti-mouse CD24 Antibody, clone M1-69</td>
			<td>Biolegend</td>
			<td>101825</td>
			<td>For flow cytometry staining of neurons and astrocytes</td>
		</tr>
		<tr>
			<td>Brilliant Violet 605 anti-mouse CD45 Antibody, clone 30-F11</td>
			<td>Biolegend</td>
			<td>103139</td>
			<td>For flow cytometry staining of microglia</td>
		</tr>
		<tr>
			<td>Brilliant Violet 785 anti-mouse/human CD11b Antibody, clone M1/70</td>
			<td>Biolegend</td>
			<td>101243</td>
			<td>For flow cytometry staining of microglia</td>
		</tr>
		<tr>
			<td>BSA Fraction V</td>
			<td>Sigma</td>
			<td>A3059-10G</td>
			<td>For SD Inhibitor</td>
		</tr>
		<tr>
			<td>CNP</td>
			<td>Abcam</td>
			<td>AB6319</td>
			<td>Mature oligodendrocytes</td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>VWR</td>
			<td>631-0149</td>
			<td>To plate out cells for microscopy</td>
		</tr>
		<tr>
			<td>Dissection Scissors</td>
			<td>Sigma</td>
			<td>S3146-1EA</td>
			<td>For dissection</td>
		</tr>
		<tr>
			<td>DMEM High glucose, sodium pyruvate, L-Glutamine</td>
			<td>Gibco</td>
			<td>21969-035</td>
			<td>For DM+/-, and for plating media</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Thermofisher</td>
			<td>18047019</td>
			<td>For SD Inhibitor, can use this or the other Dnase from sigma</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>D4263</td>
			<td>For SD Inhibitor, can use this or the other Dnase from thermofisher</td>
		</tr>
		<tr>
			<td>eBioscience Fixable Viability Dye eFluor 780</td>
			<td>Thermofisher</td>
			<td>65-0865-14</td>
			<td>Live / Dead stain</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Dumont</td>
			<td>0102-SS135-PO</td>
			<td>For dissection</td>
		</tr>
		<tr>
			<td>GFAP</td>
			<td>Invitrogen</td>
			<td>13-0300</td>
			<td>Astrocytes</td>
		</tr>
		<tr>
			<td>HBSS w Ca Mg</td>
			<td>Sigma</td>
			<td>H9269-500ML</td>
			<td>For plating media</td>
		</tr>
		<tr>
			<td>HBSS w/o Ca Mg</td>
			<td>Sigma</td>
			<td>H9394-500ML</td>
			<td>For brains to be added to</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Gibco</td>
			<td>26050-070</td>
			<td>For plating media</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma</td>
			<td>H0396</td>
			<td>For DM+/-</td>
		</tr>
		<tr>
			<td>Iba1</td>
			<td>Alpha-Laboratories</td>
			<td>019-1971</td>
			<td>Microglia</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I1882</td>
			<td>For DM+</td>
		</tr>
		<tr>
			<td>Leibovitz L-15</td>
			<td>GIbco</td>
			<td>11415-049</td>
			<td>For SD Inhibitor</td>
		</tr>
		<tr>
			<td>MBP</td>
			<td>Bio-Rad</td>
			<td>MCA409S</td>
			<td>Myelin</td>
		</tr>
		<tr>
			<td>Mouse CCL5/RANTES DuoSet ELISA Kit</td>
			<td>BioTechne</td>
			<td>DY478-05</td>
			<td>ELISA kit for quantifying concentration of CCL5 in supernatants of 96 well plate</td>
		</tr>
		<tr>
			<td>N1 media supplement</td>
			<td>Sigma</td>
			<td>N6530-5ML</td>
			<td>For DM+/-</td>
		</tr>
		<tr>
			<td>Nestin</td>
			<td>Merck</td>
			<td>MAB353</td>
			<td>Neuronal stem/progenitor cells</td>
		</tr>
		<tr>
			<td>NeuN</td>
			<td>Thermofisher</td>
			<td>PA578499</td>
			<td>Neuronal cell body</td>
		</tr>
		<tr>
			<td>NG2</td>
			<td>Sigma</td>
			<td>AB5320</td>
			<td>Immature oligodendrocytes</td>
		</tr>
		<tr>
			<td>O4 Antibody, anti-human/mouse/rat, APC</td>
			<td>Miltenyi</td>
			<td>130-119-155</td>
			<td>For flow cytometry staining of oligodendrocytes</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Sigma</td>
			<td>P0781-100ML</td>
			<td>For DM+/-, and for plating media</td>
		</tr>
		<tr>
			<td>Poly-L-Lysinehydrobromide</td>
			<td>Sigma</td>
			<td>P1274</td>
			<td>For Boric acid / poly-L-lysine solution to coat coverslips</td>
		</tr>
		<tr>
			<td>SMI31</td>
			<td>BioLegend</td>
			<td>801601</td>
			<td>Axons</td>
		</tr>
		<tr>
			<td>Sodium Tetraborate</td>
			<td>Sigma</td>
			<td>221732-100G</td>
			<td>For boric acid buffer</td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Thermofisher</td>
			<td>15596026</td>
			<td>For lysing cells for RT-qPCR</td>
		</tr>
		<tr>
			<td>Trypsin inhibitor from soybean</td>
			<td>Sigma</td>
			<td>T9003-100MG</td>
			<td>For SD Inhibitor</td>
		</tr>
	</tbody>
</table>

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  

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

The ultimate research question of my project is to figure out how we can manipulate the innate immune responses of the central nervous system to protect against viral infections. Currently, we research their response to the profoundly antiviral protein interferon beta. An experimental challenge is generating enough cells to allow for detailed investigation.Each result needs to be validated using multiple techniques and this can, unfortunately, result in many animals being culled. Due to the large number of cells generated by this novel culture technique, many different experimental conditions can be investigated from one pregnant mouse. Additionally, using in vitro alternatives, where possible, also greatly decreases animal suffering.This method reduces and refines animal use, closely following the principles of the three Rs.This culture method can be used to address many different research questions using a variety of readout methods such as ELISA, qPCR, microscopy and flow cytometry. This technique could help other researchers to reduce and refine their animal usage. We hope to improve our understanding of the innate antiviral response, modulating it to protect against opportunistic viruses such as human polyomavirus 2, or John Cunningham virus, as it is commonly known, the primary agent of PML which can be a severe side effect of immune suppressive drugs used to treat MS and other diseases.

Microscopy 
Cultures grown on glass coverslips are ideal to analyze by microscopy. To visualize the development of the cultures, the coverslips were fixed in 4% paraformaldehyde (PFA) at multiple timepoints from DIV0 (once cells were attached) until DIV28. The cultures were stained for immunofluorescence imaging as previously described5 using three different staining combinations: NG2 (immature oligodendrocytes) and nestin (neuronal stem/progenitor cells) as developmental mar...

Watch this Scientific Journal Video about Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity at JoVE.com

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 ...

establishing-mixed-neuronal-glial-cell-cultures-from-embryonic-mouse

Research

JoVE Journal

Neuroscience

Murine E17 Embryonal Brain Dissection, Cell Plating, and Maintenance

Begin by placing a sacrificed pregnant mouse on its back on a dissection board. Using forceps, pinch the abdominal midline. With a pair of sharp scissors, cut the abdomen through the skin and the peritoneum from the genitalia to the rib cage over the midline.Remove the uterus containing the embryos and place it immediately on ice. Carefully cut through the yolk sac on the placental sides and remove the embryos. Then, place the decapitated embryonic heads into an iced dish containing calcium and magnesium-deficient HBSS.Place the head into a 35-millimeter dish facing left. Pierce one eye with the edge of the forceps, firmly holding the chin with the other. Gently tear the scalp skin from the nape along the midline towards the snout.Use the angled forceps to enter through the white oval spinal cord and crack the skull open along the midline, exposing the brain. Gently peel the skull away from the sides. Then, lift the brain out of the skull and dispose of the skull.Use forceps to remove the meninges. Place the brains in a bijou containing two milliliters of calcium and magnesium-deficient HBSS. Add 250 microliters of 10X trypsin to the bijou.Triturate the brains by shaking the bijou and then incubate for 15 minutes at 37 degrees Celsius. Next, add two milliliters of soybean trypsin inhibitor to each bijou. Shake to disperse the inhibitor evenly.Without centrifuging, transfer two milliliters of the supernatant from each bijou into a 15-milliliter centrifuge tube. Using a 19-gauge needle attached to a five-milliliter syringe, aspirate the suspension twice to triturate the remaining cells in the bijou. Repeat aspiration twice with a 21-gauge needle.Using a 23-G needle, transfer the cells from the bijou into the 15-milliliter centrifuge tube containing the cell supernatant and centrifuge at 200 G for five minutes at room temperature. Using a five-millimeter serological pipette, transfer all the supernatant into a new 15-milliliter centrifuge tube without disturbing the pellet. Repeat centrifugation.Add 10 milliliters of the plating media to a tube containing the combined pellets from both centrifugation steps and mix well to create a whole suspension. Stain the cells with trypan blue and count using a hemocytometer or a cell counter. Plate the cells by adding the required volume of the murine E17 embryonic brain cell suspension into a well plate.Incubate the cells at 37 degrees Celsius under 5 to 7%carbon dioxide for two to four hours. After removing the media, add new differentiation media to each well. Press down any floating cover slips using a sterile pipette tip.Maintain cultures by removing part of the supernatant and replacing it with fresh differentiation media three times each week. Cultures were visualized by immunofluorescence staining for NG2 and nestin as developmental markers, SMI31, MBP, and NeuN as neuronal markers, and CNP, GFAP and Iba1 as glial markers Infection of cultures with neurotropic Semliki Forest virus affected the oligodendrocytes and the neurons. qRT-PCR investigations of the infected cells demonstrated upregulation of the chemotactic cytokine Ccl5 mRNA in cultures treated with interferon beta.ELISA studies displayed increased Ccl5 in the supernatant, thus displaying a correlation between mRNA and protein expression. Flow cytometric studies of single-cell suspension showed that 70%of the cells remained viable. A large number of microglia, neurons, and astrocytes were present, while oligodendrocytes were the least abundant.

1.6K Views.  University of Glasgow.  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.

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

Summary