We would like to thank members of the Tsirka lab for their advice and helpful comments. This work was supported by R01NS42168 to SET, 12PRE12060489 to RB, an NSF-3MT IGERT and a Turner Dissertation fellowship to LT, and NSF-3MT IGERT to JCN.

1. Dissection (Day 0)
<ol>
	<li>Induce anesthesia to p0-2 mouse pups with hypothermia. Wipe the heads of the pups with a Kimwipe soaked in 70% ethanol to disinfect the tissue.</li>
	<li>Remove the heads with a pair of scissors, using 4 pups per 10 cm culture plate. Place the heads in a petri dish containing ice-cold Hank's buffer.</li>
	<li>Anchor the heads using curved forceps through the eye sockets and carefully remove the skin covering the skull with straight microforceps.</li>
	<li>Remove the cranial bones with straight microforceps, using caution to not puncture or damage the cortices. The most effective way is to begin the removal starting near the cerebellum, which is located at the base of the head, as this region will be discarded.</li>
	<li>Remove the brain with a small spatula, and place the (4) brains in a fresh petri dish containing ice-cold Hank's buffer.</li>
	<li>All steps from this point on utilize microforceps and are performed under a dissection microscope. Starting on the ventral side of the brain, anchor the tissue by holding the cerebellum and make two small incisions on either side of the midbrain. Be careful not to cut all the way through the tissue, as the cortices cover the midbrain in this region.</li>
	<li>Gently tease the midbrain and cerebellum in one piece from the two cortices. The two cortices should form a concave shape.</li>
	<li>Separate the two cortices and orient one single cortex with the medial side up for further dissection. The hippocampus can be difficult to observe, but it is located opposite of the olfactory bulb which appears as a small nodule on the pointed end of the cortex.</li>
	<li>Remove the hippocampus, which has a crescent-shape. Flip the cortex over to view the dorsal side.</li>
	<li>Using the olfactory bulb as a starting point, remove all meninges and the olfactory bulb itself from the cortex.</li>
	<li>The dissected cortices should be placed into 15 ml conical tubes with 14 ml of cold Hank's buffered saline on ice.</li>
</ol>
2. Cell Culture (Day 0)
<ol>
	<li>Pre-coat 10 cm tissue culture dishes with 5 &mu;g/ml of Poly-D-Lysine (PDL) diluted in autoclaved water for 3 hr at 37 &deg;C.</li>
	<li>Aspirate PDL, and wash plates once with autoclaved water. Dry plate under the UV in the tissue culture hood for 20 min. This step should be performed just prior to the dissection process.</li>
	<li>Aspirate Hank's buffer from tubes containing the cortices from 4 brains each, apply 4 ml of 1x trypsin/EDTA solution, triturate tissue with p1000 tip, and place tubes in 37 &deg;C incubator for 15 min.</li>
	<li>Add 4 ml of complete microglial media per tube to stop the enzymatic digestion, mix, and spin down contents at 1.5K rpm for 5 min.</li>
	<li>Aspirate supernatant and repeat wash with 4 ml of complete media. Resuspend cells in 10 ml of complete microglial media (DMEM with 10% FBS, 1% sodium pyruvate, 0.08% gentamycin), and filter through a 40 micron mesh cell strainer.</li>
	<li>Plate at a density of 8 cortices per 10 ml in a 10 cm tissue culture plate and place in a tissue culture incubator at 37 &deg;C and 5% CO2 (See Adult Microglia protocol).</li>
</ol>
(Day 3)
<ol>
	<li>Change media in all cell culture dishes with complete microglial media.</li>
</ol>
(Day 10)
<ol>
	<li>Add 400 microliters of 60 mM Lidocaine in HBSS (to detach microglia) into medium of 10-cm tissue culture plates and incubate at room temperature (RT) for 10-15 min.</li>
	<li>Collect the media/cell suspension from the plate, and wash the plate once with Hank's buffer. Collect the wash buffer to recover remaining microglia.</li>
	<li>Add 5 mM EDTA (pH 8.0) to the cell suspension to a final concentration of 50 &mu;M or at a 1/100 dilution.</li>
	<li>Spin down cell suspension at 1,000 x g (1,500 rpm) for 5 min and re-suspend in 1 ml DMEM with 1% FBS.</li>
	<li>Count viable cell number (as per Adult Microglia 3.1/3.2) and split cells into tissue culture plates at the desired experimental density. Approximately 1x106 microglia are harvested from a 10 cm plate containing the cortices from 4 brains. For immunofluorescence, a density of 2.5x104 cells per 18 mm coverslip is recommended.</li>
	<li>Allow at least 24 hr for microglial cells to fully return to their ramified, resting state prior to use.</li>
</ol>
Protocol Text: (Adult Microglia)
1. Tissue Collection
<ol>
	<li>Coat tissue culture plates with Poly-D-Lysine (PDL) for 3 hr at 37 &deg;C or overnight at 4 &deg;C. Wash the plates once with autoclaved water immediately prior to use.</li>
	<li>Euthanize mouse by CO2 asphyxiation and ascertain that euthanasia is complete. Clean spinal cord area with 70% ethanol.</li>
	<li>Using a pair of scissors cut the skin on top of the spinal cord; then proceed to cut the spinal cord out from region T1 to T12. Cut the remaining muscle off of the sides of the spinal cord.</li>
	<li>Slowly cut the vertebrae using microscissors as you gently hold the spinal cord with your fingertips. Be careful not to puncture the spinal cord as the tissue is very soft. Slowly extract the spinal cord using microforceps.</li>
	<li>Submerge the spinal cord in a petri dish containing ice-cold HBSS. Using a light microscope remove all visible meninges using microforceps</li>
	<li>Cut the spinal cord into transversal segments small enough to generate several fragments. The thickness of the fragments should not be more than 2 mm in order to facilitate efficient digestion. Transfer the spinal cord pieces to a 15 ml conical tube containing 1 ml ice-cold HBSS. Keep the tube on ice until the digestion step.</li>
</ol>
2. Digestion of Tissue
<ol>
	<li>Aspirate HBSS from 15 ml conical tube containing spinal cord tissue. Be careful not to aspirate any piece of tissue.</li>
	<li>Add 1 ml of trypsin (2.5 g/L irradiated porcine trypsin and 0.2 g/L EDTA in HBSS) and incubate at 37 &deg;C for 30 min.</li>
	<li>To stop the digestion remove the trypsin and add 3 ml of primary microglia medium which contains serum to halt the enzymatic digestion.</li>
	<li>Pipet up and down to dislodge the tissue. Filter the cell suspension using a 40 &mu;m cell strainer.</li>
</ol>
3. Cell Counting and Plating
<ol>
	<li>Mix equal volume of cell suspension and DAPI solution.</li>
	<li>Count cells using a hemocytometer.</li>
	<li>Plate at a density between 5-7x105 cells/ml in PDL-coated 35 x 10 mm dishes . Plating at a lower density prevented cell growth even when cells were cultured in 12 well plates which have a smaller area compared to 35 x 10 mm dishes.</li>
</ol>

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Authors have nothing to disclose.

Microglia modulate CNS normal functioning as well as inflammatory responses to various pathologies. Functional synaptic remodeling by microglia has been implicated in the maintenance of normal brain homeostasis 15. During the neurogenic cascade they participate in the clearance of neural progenitor cells from the dentate gyrus of the hippocampus 4, 16. Therefore, it is necessary to develop a culture system in which to study neonatal and adult microglia, which will cover the vast swath of deve...

Microglia are the resident immune cells of the CNS, most closely resembling peripheral macrophages in structure and function 1. It has recently been demonstrated that postnatal microglial cells derive from primitive myeloid progenitors and are generated before the eighth day of embryogenesis, upending the previous notion that postnatal hematopoietic progenitors are the source of microglia in the adult brain 2. They play key roles in several neurological diseases and can quickly respond to infection or injury by releasing pro-inflammatory or anti-inflammatory cytokines 3. Thus microglia encompass a standalone unit in the CNS that can be manipulated to affect disease progression. Developing robust and reproducible methods to isolate and culture neonatal or adult microglial cells is important to future studies.
Microglia are known to be critical players in a number of brain pathologies. More recently, roles are emerging for the cells in normal brain development and function as microglia phagocytose excess neural progenitor cells from the dentate gyrus of the hippocampus 1, 4. Microglia can also modulate several neurological conditions that affect the spinal cord, such as MS, neuropathic pain, and spinal cord injury 5-7. Spinal cord microglia react differently compared to brain microglia in response to activation signals 8, 9, probably due to differences in the local environment. Thus it is important to establish an appropriate in vitro system to culture and study spinal cord microglia. Neonatal microglia produce significantly more of the pro-inflammatory cytokine nitric oxide compared to adult cells after in vitro stimulation with IFN-&gamma; or TNF-a10,11 further highlighting the need to use adult cells to study microglia in the context of certain diseases.
The protocol we employ in the lab to culture neonatal microglia is a variation of recent methods which utilize shaking of mixed glial cultures in an effort to remove the microglia from the surface of the cell culture flask 12. We also describe a method to culture microglia from the adult mouse spinal cord based on a protocol first described by Yip, et al13. This method provides a quicker way to culture adult cells compared to other available protocols 14. The resulting preparation is 70% microglia; the remaining percentage is composed of astrocytes. Although the purity of our culture is lower compared to other published methods 13, this culture system is useful for exploring the microglial in culture response to various activating stimuli as well as for the study of diseases that mainly affect the spinal cord and in which a strong inflammatory response is a main feature.
All the protocols described have been approved by the Stony Brook University IACUC.

<table><tbody><tr><td>EDTA, 5mM</td><td>Invitrogen</td><td>15567-028</td><td></td></tr><tr><td>Lidocaine, 60mM</td><td>Sigma</td><td>L-5647</td><td></td></tr><tr><td>Trypan Blue</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Cellgro</td><td>25-052-CI</td><td></td></tr><tr><td>DMEM, 1X</td><td>Cellgro</td><td>10-017</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Cellgro</td><td>25-000-Cl</td><td></td></tr><tr><td>Gentamycin Sulfate</td><td>Biowittaker</td><td>17-518Z</td><td></td></tr><tr><td>35 x 10mm tissue culture dish</td><td>Falcon</td><td>353001</td><td></td></tr><tr><td>Poly-D-Lysine, 100 &amp;mu;g/ml</td><td></td><td></td><td>Dilute 1:20</td></tr><tr><td>HBSS, 1X</td><td>Cellgro</td><td>20-023-CV</td><td></td></tr></tbody></table>

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.

An example of resting and activated microglial cells is shown in Figure 1. The microglia were visualized 24 hr after plating (Figure 1a) and exhibit ramified (resting) morphology. Exposure to the priming reagent, bacterial lipopolysaccharide (LPS) results in changes in microglial morphology as the cells become activated (Figure 1b).
An example of counting the cells for plating is shown in Figure 2. Due to the presence of cell ...

Watch this Scientific Journal Video about Culturing Microglia from the Neonatal and Adult Central Nervous System at JoVE.com

Culturing Microglia from the Neonatal and Adult Central Nervous System

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 ...

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Immunology and Infection

27.8K Views.  Stony Brook University. 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.

Video: Culturing Microglia from the Neonatal and Adult Central Nervous System

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

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 ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Neuroscience

Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during development, homeostasis, and disease. Although macrophages have been studied in detail for decades, specialized features of microglia, the tissue-resident macrophages of the CNS, have remained largely mysterious, in part due to limitations in the ability to recapitulate mature microglial properties in culture. Here, we illustrate a straightforward procedure for the rapid isolation of pure microglia from the mature rodent brain. We also describe serum-free culture conditions that support high levels of microglial viability over time. Microglia cultured under these defined-medium conditions exhibit elaborate ramified processes and dynamic surveillance behavior. We illustrate some effects of serum exposure on cultured microglia and discuss how these serum-free cultures compare to both serum-exposed cultures as well as microglia in vivo.

Efforts to understand microglial function in detail have been hindered by the lack of microglial culture models that recapitulate the properties of mature in vivo microglia. This protocol describes an isolation and culture approach designed to maintain robust survival of highly ramified mature rat microglia under defined-medium conditions.

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

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 ...

Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. Microglia can adopt a large spectrum of activation phenotypes. Moreover, microglia that endorse pro-inflammatory phenotype is associated with both neurodevelopmental and neurodegenerative disorders. In vitro studies are widely used in research to evaluate potential therapeutic strategies in specific cell types. In this context studying microglial activation and neuroinflammation in vitro using primary microglial cultures is more relevant than microglial cell lines or stem-cell-derived microglia. However, the use of some primary cultures might suffer from a lack of reproducibility. This protocol proposes a reproducible and relevant method of magnetically isolating microglia from neonate pups. Microglial activation using several stimuli after 4 h and 24 h by mRNA expression quantification and a Cy3-bead phagocytic assay is demonstrated here. The current work is expected to provide an easily reproducible technique for isolating physiologically relevant microglia from juvenile developmental stages.

Primary microglia cultures are commonly used to evaluate new anti-inflammatory molecules. The present protocol describes a reproducible and relevant method to magnetically isolate microglia from neonate pups.

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. ...

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

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

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

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

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