The study was supported by Tongji Hospital (HUST) Foundation for Excellent Young Scientist (Grant No. 2020YQ06).

All the animal procedures have been approved by the Institute of Animal Care Committee of Tongji Medical College, Huazhong University of Science and Technology, China.
1. Materials
<ol>
	<li>Prepare the following solutions before beginning the protocol.
	<ol>
		<li>Prepare the loading buffer by adding fetal bovine serum (FBS, 2%) to phosphate buffered saline (PBS).</li>
		<li>Add neutral red (NR) dye (final 1%) to PBS.</li>
	</ol>
	</li>
	<li>Prepare the following solutions using the commercially available Adult Brain Dissociation Kit (see the Table of Materials).
	<ol>
		<li>To prepare enzyme mix 1, pipette 1.9 mL of Buffer Z and 50 &#181;L of Enzyme P into a 15 mL centrifuge tube.</li>
		<li>To prepare enzyme mix 2, pipette 20 &#181;L of Buffer Y and 10 &#181;L of Enzyme A into a 1.5 mL microcentrifuge tube.</li>
		<li>Prepare the debris removal solution.</li>
	</ol>
	</li>
</ol>
2. Mice perfusion and dissection
<ol>
	<li>Intraperitoneally (i.p.) inject the mouse with 500 &#181;L of 1% NR dye in PBS 2-3 h before anesthesia. 
	NOTE: Intraperitoneal injection of NR in demyelinating mouse models helps discern the lesions (Figure 1).</li>
	<li>Anesthetize the mouse with pentobarbital (50 mg/kg, i.p.), and ensure that the mouse is successfully anesthetized by examining the pain responses.</li>
	<li>Open the thoracic cavity of the mouse and expose the heart.</li>
	<li>Cut off a small corner of the right atrium carefully and intracardially perfuse the mouse with 20-30 mL of cold PBS from the left ventricle.</li>
	<li>Decapitate the mouse under anesthesia.</li>
	<li>Open the skull of the mouse and carefully free the brain from the skull.</li>
	<li>Transfer the brain to the mouse brain slice mold and cut the brain into 0.1 cm slices.</li>
	<li>Remove the brain slices and place them in cold PBS in a Petri dish (60/15 mm). Microdissect the lesions labeled by NR dye around the corpus callosum under a stereomicroscope using microsurgical forceps. 
	NOTE: Ensure that the dissected tissue is as complete as possible to facilitate the subsequent transfer; the total dissection progress should be completed in 2 h.</li>
	<li>Transfer the dissected tissue to 15 mL centrifuge tubes containing the appropriate amount of cold loading buffer. 
	&#8203;NOTE: Pool the corpus callosum tissue from 2-3 mice together in one tube as one sample.</li>
</ol>
3. Tissue dissociation
<ol>
	<li>Centrifuge the dissected tissue at 300 &#215;g&#160;for 30 s (after reaching this speed) to collect the sample at the bottom of the tube.</li>
	<li>Preheat enzyme mix 1 and enzyme mix 2 to 37 &#176;C in an incubator.</li>
	<li>Add 1,950 &#181;L of preheated enzyme mix 1 to one sample and digest in an incubator at 37 &#176;C for 5 min.</li>
	<li>Add 30 &#181;L of preheated enzyme mix 2 and mix gently.</li>
	<li>Digest in an incubator at 37 &#176;C for 15 min and mix gently every 5 min.</li>
	<li>Add 4 mL of cold PBS to the tube after digestion and shake gently.</li>
	<li>Place 70 &#181;m filters on 50 mL centrifuge tubes and prewet the filters with 500 &#181;L of cold PBS. Filter the dissociated tissue by passing the digested tissue samples through the filters, and transfer the filtered suspension in the 50 mL centrifuge tube to a 15 mL centrifuge tube. 
	NOTE: Skip this step if the amount of tissue is too small.</li>
	<li>Centrifuge the filtered tissue samples at 300 &#215;g for 10 min at 4 &#176;C and aspirate the supernatant slowly and completely. 
	NOTE: All the steps should be performed on ice except for the enzymatic digestion.</li>
</ol>
4. Debris removal
<ol>
	<li>Resuspend the cell pellet gently with 1,550 &#181;L of cold PBS.</li>
	<li>Add 450 &#181;L of cold solution for debris removal and mix well.</li>
	<li>Overlay the above mixture very slowly and gently with 2 x 1 mL of cold PBS using a 1,000 &#181;L pipette. 
	NOTE: Tilt the centrifuge tube by 45 &#176; and slowly add PBS along the tube wall with the pipette.</li>
	<li>Transfer the tube slowly and gently to a centrifuge and spin at 4 &#176;C and 3,000 &#215; g for 10 min.</li>
	<li>Look for three layers after centrifugation. Aspirate the two top layers completely by using a 1,000 &#181;L pipette (Figure 2).</li>
	<li>Fill the tube up to 5 mL with cold loading buffer and gently invert the tube three times.</li>
	<li>Centrifuge at 4 &#176;C and 1,000 &#215; g for 10 min. Aspirate the supernatant completely and avoid disrupting the cell pellet.</li>
</ol>
5. Magnetic separation of microglial cells
<ol>
	<li>Resuspend the cell pellet with 90 &#181;L of loading buffer and add 10 &#181;L of CD11b (Microglia) beads.</li>
	<li>Mix well and incubate for 15 min at 4 &#176;C.</li>
	<li>Add 1 mL of loading buffer and pipette the liquid gently up and down with a 1,000 &#181;L pipette to wash the cells after the incubation. Centrifuge the cells at 4 &#176;C and 300 &#215;&#160;g for 10 min and aspirate the supernatant completely to remove the unbound beads.</li>
	<li>Resuspend the cells in 500 &#181;L of loading buffer.</li>
	<li>Place the MS column with its separator for positive selection in the magnetic field.</li>
	<li>Rinse the column with 500 &#181;L of loading buffer to protect the cells and ensure the efficiency of magnetic sorting based on the protocol of the manufacturer.</li>
	<li>Apply the cell suspension onto the MS column and discard the flowthrough containing unlabeled cells.</li>
	<li>Add 500 &#181;L of loading buffer to the column three times to wash away cells that adhere to the column wall and discard the flowthrough. 
	NOTE: Only add new loading buffer for washing when the column reservoir is completely empty.</li>
	<li>Remove the column from the separator after washing the column and place it on a 15 mL centrifuge tube.</li>
	<li>Add 1 mL of loading buffer into the column and push the plunger to the bottom of the column to flush out the magnetically labeled cells. Repeat this step three times to completely collect the magnetically labeled cells.</li>
</ol>

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The authors have declared that no competing interests exist.

The protocol proposes a method to isolate microglia around the demyelinating lesions, which can help study the functional characteristics of microglia in inflammatory demyelinating diseases. Microglia captured using CD11b beads exhibit high purity and viability. Critical steps in the protocol include the precise localization of foci and optimal microglial purification. In protocol step 2.1, it is necessary to inject the NR solution 2 h before sacrificing the mouse to ensure that the lesions can be accurately displayed<su...

Microglia originate from yolk-sac progenitors, which reach the embryonic brain very early and participate in the development of the CNS1,2. For instance, they are involved in synaptic pruning3 and regulating axonal growth4. They secrete factors that promote neuronal survival and help neuronal localization5. At the same time, they are involved in removing abnormal cells and apoptotic cells to ensure normal brain development6. In addition, as the immune-competent cells of the brain, microglia continually surveil the brain parenchyma to clear dead cells, dysfunctional synapses, and cellular debris7. It has been demonstrated that microglial activation plays an important role in a variety of diseases, including inflammatory demyelinating diseases, neurodegenerative diseases, and brain tumors. Activated microglia in multiple sclerosis (MS) contribute to the differentiation of oligodendrocyte precursor cells (OPCs) and regeneration of myelin by engulfing myelin debris8.
In Alzheimer&#39;s disease (AD), accumulation of amyloid beta (A&#946;) activates microglia, which affects the phagocytic and inflammatory functions of microglia9. Activated microglia in the glioma tissue, called glioma-associated microglia (GAM), can regulate the progression of glioma and ultimately affect the prognosis of patients10. The activation profoundly alters the microglial transcriptome, resulting in morphological changes, expression of immune receptors, increased phagocytic activity, and enhanced cytokine secretion11. There are different subsets of activated microglia in neurodegenerative diseases such as disease-associated microglia (DAM), activated response microglia (ARM), and microglial neurodegenerative phenotype (MGnD)8.
Similarly, multiple dynamic functional subsets of microglia also coexist in the brain in inflammatory demyelinating diseases12. Understanding the heterogeneity between different subsets of microglia is essential to investigate the pathogenesis of inflammatory demyelinating diseases and to find their potential therapeutic strategies. The heterogeneity of microglia mainly depends on the molecular specificity8. It is essential to describe the molecular alterations of microglia accurately for the study of the heterogeneity. Advances in single-cell RNA-sequencing (RNA-seq) technology have enabled the identification of the molecular characteristics of activated microglia in pathological conditions13. Therefore, the ability to isolate cell populations is critical for further investigation of these target cells under specific conditions.
Studies performed to understand the characteristics and functions of microglia are usually in vitro studies, since it has been found that large numbers of primary microglia can be prepared and cultured from mouse pup brains (1-3 days old), which attach to the culture flasks and grow on the plastic surface with other mixed glial cells. Subsequently, pure microglia can be isolated based on the different adhesiveness of mixed glial cells14,15. However, this method can only isolate microglia from the perinatal brain and takes several weeks. Potential variables in cell culture may influence microglial characteristics such as molecular expression16. Moreover, microglia isolated by these methods can only participate in in vitro experiments by simulating the conditions of CNS diseases and cannot represent the characteristics and functions of microglia in in vivo disease states. Therefore, it is necessary to develop methods for isolating microglia from the adult mouse brain.
Fluorescence-activated cell sorting (FACS) and magnetic separation are two widely used methods, although they have their own different limitations16,17,18,19. Their respective advantages and disadvantages will be contrasted in the discussion section. The maturation of MACS technology offers the possibility to rapidly purify cells. Huang et al. have developed a convenient method to label demyelinating lesions in brains20. Combining these two technical approaches, we propose a rapid and efficient columnar CD11b magnetic separation protocol, providing a step-by-step description to isolate microglia around demyelinating lesions in adult mouse brains and preserve the molecular characteristics of microglia. The focal demyelinating lesions were caused by the stereotactic injection of 2 &#181;L of lysolecithin solution (1% LPC in 0.9% NaCl) in the corpus callosum 3 days before starting the protocol21. This protocol lays the foundation to perform the next step in in vitro experiments. Moreover, this protocol saves time and remains feasible for widespread use in various experiments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL Micro Centrifuge Tubes</td>
			<td>BIOFIL</td>
			<td>CFT001015</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Centrifuge Tubes</td>
			<td>BIOFIL</td>
			<td>CFT011150</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tubes</td>
			<td>BIOFIL</td>
			<td>CFT011500</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m Filter</td>
			<td>Miltenyi Biotec</td>
			<td>130-095-823</td>
			<td></td>
		</tr>
		<tr>
			<td>Adult Brain Dissociation Kit, mouse and rat</td>
			<td>Miltenyi Biotec</td>
			<td>130-107-677</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J Mice</td>
			<td>SJA Labs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b (Microglia) Beads, human and mouse</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-634</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>BOSTER</td>
			<td>PYG0001</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>BD Biosciences</td>
			<td>V10</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr>
			<td>MS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral Red</td>
			<td>Sigma-Aldrich</td>
			<td>1013690025</td>
			<td></td>
		</tr>
		<tr>
			<td>NovoCyte Flow Cytometer</td>
			<td>Agilent</td>
			<td></td>
			<td>A system consisting of various parts</td>
		</tr>
		<tr>
			<td>NovoExpress</td>
			<td>Agilent</td>
			<td>1.4.1</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>BOSTER</td>
			<td>PYG0021</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital</td>
			<td>Sigma-Aldrich</td>
			<td>P-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>MshOt</td>
			<td>MZ62</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of mouse primary microglia by magnetic-activated cell sorting in animal models of demyelination

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). Microglia can be divided into resting state and activated state and can rapidly change state in response to the microenvironment of the brain. Microglia will be activated under different pathological conditions and exhibit different phenotypes. In addition, there are many different subgroups of activated microglia and great heterogeneity between different subgroups. The heterogeneity mainly depends on the molecular specificity of microglia. Studies have revealed that microglia will be activated and play an important role in the pathological process of inflammatory demyelination. To better understand the characteristics of microglia in inflammatory demyelinating diseases such as multiple sclerosis and neuromyelitis optica spectrum disorder, we propose a perilesional primary microglial sorting protocol. This protocol utilizes columnar magnetic-activated cell sorting (MACS) to obtain highly purified primary microglia and preserve the molecular characteristics of microglia to investigate the potential effects of microglia in inflammatory demyelinating diseases.

Microglia isolated using CD11b beads have high purity 
Microglial cells around the lesions in demyelination mouse models were isolated using the above-mentioned protocol and tested by flow cytometry. Cells are fluorescently labeled with CD11b-fluorescein isothiocyanate (FITC) and CD45-allophycocyanin (APC) to determine microglia in flow cytometry according to the manufacturer&#39;s instructions. There are multiple literatures demonstrating that CD11b and CD45 antibodies are enough to check for the p...

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Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination

Here, we present a protocol to isolate and purify primary microglia in animal models of demyelinating diseases, utilizing columnar magnetic-activated cell sorting.

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). ...

isolation-mouse-primary-microglia-magnetic-activated-cell-sorting

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3.4K Views.  Huazhong University of Science and Technology. Here, we present a protocol to isolate and purify primary microglia in animal models of demyelinating diseases, utilizing columnar magnetic-activated cell sorting.

Video: Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination

Mouse Models of Periventricular Leukomalacia

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature ...

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

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

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

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

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

Primary Cell Culture of Purified GABAergic or Glutamatergic Neurons Established through Fluorescence-activated Cell Sorting

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can be cultured in defined media for 16 days in vitro and are amenable to any analyses typically performed on dissociated cultures, including electrophysiological, morphological and survival analyses. The major advantage of these cultures is that specific cell types can be selectively studied in the absence of complex external influences, such as those arising from glial cells or other neuron types. When planning experiments with purified cells, however, it is important to note that neurons strongly depend on glia-conditioned media for their growth and survival. In addition, glutamatergic neurons further depend on glia-secreted factors for the establishment of synaptic transmission. We therefore also describe a method for co-culturing neurons and glial cells in a non-contact arrangement. Using these methods, we have identified major differences between the development of GABAergic and glutamatergic neuronal networks. Thus, studying cultures of purified neurons has great potential for furthering our understanding of how the nervous system develops and functions. Moreover, purified cultures may be useful for investigating the direct action of pharmacological agents, growth factors or for exploring the consequences of genetic manipulations on specific cell types. As more and more transgenic animals become available, labeling additional specific cell types of interest, we expect that the protocols described here will grow in their applicability and potential.

This protocol describes a cell sorting based method for the purification and culture of fluorescent GABAergic or glutamatergic neurons from the neocortex and hippocampus of postnatal mice or rats.

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy

Transgenic mouse models have proved to be powerful tools in studying various aspects of human neurological disorders, including epilepsy. The SCN1A-associated genetic epilepsies comprise a wide spectrum of seizure disorders with incomplete penetrance and clinical variability. SCN1A mutations can result in a large variety of seizure phenotype ranging from simple, self-limited fever-associated febrile seizures (FS), moderate-level genetic epilepsy with febrile seizures plus (GEFS+) to more severe Dravet Syndrome (DS). Although FS are commonly seen in children below 6-7 years of age who do not have genetic epilepsy, FS in GEFS+ patients continue to occur into adulthood. Traditionally, experimental FS have been induced in mice by exposing the animal to a stream of dry air or heating lamps, and the rate of change in body temperature is often not well controlled. Here, we describe a custom-built heating chamber, with a plexiglass front, that is fitted with a digital temperature controller and a heater-equipped electric fan, which can send heated forced air into the test arena in a temperature-controlled manner. The body temperature of a mouse placed in the chamber, monitored through a rectal probe, can be increased to 40-42 &#176;C in a reproducible manner by increasing the temperature inside the chamber. Continual visual monitoring of the animals during the heating period demonstrates induction of heat-induced seizures in mice carrying an FS mutation at a body temperature that does not elicit behavioral seizures in wild-type litter mates. Animals can be easily removed from the chamber and placed on a cooling pad to rapidly return body temperature to normal. This method provides for a simple, rapid, and reproducible screening protocol for the occurrence of heat-induced seizures in epilepsy mouse models.

The goal of the method is to screen for hyperthermia or heat-induced seizures in mouse models. The protocol describes the use of a custom-built chamber with continuous monitoring of the body temperature to determine whether elevated body temperature leads to seizures.

Transgenic mouse models have proved to be powerful tools in studying various aspects of human neurological disorders, including epilepsy. The ...

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. ...

Efficient Extraction of Astrocytes and Microglia from the Adult Mouse Spinal Cord

Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For In Vitro Assays and Transcriptomic Studies

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic cells exhibit rapid responses to environmental changes and display significant heterogeneity in terms of morphology, transcriptional profiles, and functions. While our understanding of the functions of glial cells in health and disease has advanced substantially, there remains a need for in vitro, cell-specific analyses conducted in the context of insults or injuries to comprehensively characterize distinct cell populations. Isolating cells from the adult mouse offers several advantages over cell lines or neonatal animals, as it allows for the analysis of cells under pathological conditions and at specific time points. Furthermore, focusing on spinal cord-specific isolation, excluding brain involvement, enables research into spinal cord pathologies, including experimental autoimmune encephalomyelitis, spinal cord injury, and amyotrophic lateral sclerosis. This protocol presents an efficient method for isolating astrocytes and microglia from the adult mouse spinal cord, facilitating immediate or future analysis with potential applications in functional, molecular, or proteomic downstream studies.

Author Spotlight: Isolation of Glial Cells from the Adult Mouse Spinal Cord 

This protocol outlines the isolation of purified astrocytes and microglia from the adult mouse spinal cord, facilitating subsequent applications such as RNA analysis and cell culture. It includes detailed cell dissociation methods and procedures designed to enhance both the quality and yield of isolated cells.

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic ...