Name: selective depletion of microglia from cerebellar granule cell cultures using l-leucine methyl ester
Uploaded: 2015-07-07T15:00:00.000+00:00
Duration: 8 min 10 s
Description: Watch this Scientific Journal Video about Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester at JoVE.com

This research was support by an Aims2Cure, UK and a UCL Impact Award Ph.D. studentship to JMP and an MRC Capacity Building Ph.D. studentship in Dementia to JMP.

All experiments described herein were performed in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986.
1. Preparation of Instruments, Culture Media, and Dishes
<ol>
	<li>Prepare two stainless steel laboratory dissecting scissors and two stainless steel laboratory forceps. Autoclave all instruments and place in a culture hood O/N under ultraviolet light (UV) light for sterilization.</li>
	<li>Make 500 ml of minimum essential media (MEM) culture medium (10% Foetal Bovine Serum [FBS], 20 mM KCl, 25 mM NaHCO3, 30 mM D-glucose, 2 mM L-glutamine, 100 U/ml penicillin, 100 &#181;g/ml streptomycin and 6 &#181;g/ml ampicillin) and filter sterilize. Store at 4 &#176;C for months.</li>
	<li>Prepare 24 well culture plates containing 14 mm, number 1 thickness coverslips. Autoclave the coverslips and coat in 100 mg/L poly-d-lysine (PDL) as previously described 3. UV light sterilize O/N.</li>
</ol>
2. Preparation of CGC Culture Solutions
<ol>
	<li>Prepare &#8216;solution A&#8217; (100 ml) in autoclaved, UV light treated, sterile dH2O containing 250 mg of glucose, 300 mg of Bovine Serum Albumin [BSA], 955 mg of Phosphate Buffered Saline [PBS] (Ca2+ and Mg+ free), and 1 ml of 3.8% MgSO4&#183;7H2O.</li>
	<li>Prepare &#8216;solution B&#8217; (10 ml) containing 1 ml of 5 mg/ml trypsin diluted in 9 ml of solution A.</li>
	<li>Prepare &#8216;solution C&#8217; (10 ml) containing 1,000 units DNase, 0.5 mg of soybean trypsin inhibitor, 100 &#181;l of 3.8% MgSO4&#183;7H2O, diluted to 10 ml&#160;with solution A.</li>
	<li>Prepare &#8216;solution D&#8217; (20 ml) containing 3.2 ml of solution C, diluted to 20 ml with solution A.</li>
	<li>Prepare BSA/Earle&#8217;s Balanced Salt Solution (EBSS) solution containing EBSS, 25 mM NaHCO3, 3 mM MgSO4&#183;7H2O, and 4% BSA, pH 7.4, as previously described3.</li>
	<li>Prepare LME by adding pure LME into 5 ml of MEM culture medium to give a final concentration of 150 mM LME, return the solution to pH 7.4 using a benchtop pH meter, and filter sterilize using a 28 mm polyether sulfone (PES) 0.2 &#181;m syringe filter.</li>
</ol>
3. Culturing the CGCs
<ol>
	<li>Collect cerebella from 4-7 day old rat pups as previously described3. Place immediately in Petri dish containing 5 ml solution A on ice.</li>
	<li>Pour off excess solution from cerebella and place tissue in the Petri dish lid.</li>
	<li>Chop tissue finely with a well-flamed razor blade in at least three different directions.</li>
	<li>Add chopped tissue to solution B (rinse Petri dish with solution to ensure limited tissue is lost) and place in water bath at 37 &#176;C for 5 min. Shake gently every couple of min.</li>
	<li>Add solution D (20 ml) to the tube to neutralize the trypsin, shake and centrifuge at 65 x g for 5 min.</li>
	<li>Pour off supernatant.</li>
	<li>Resuspend in 4 ml of solution C. Triturate well (approximately 10 times each) with three flamed glass pipettes of decreasing diameter, until the suspension is as homogenous as possible.</li>
	<li>Slowly and gently add a few drops of this homogenate on top of the BSA/EBSS. If it starts to sink through the BSA/EBSS, triturate more and/or add a little more solution C. The homogenate should sit in a layer on top of the BSA/EBSS. Do not shake. Spin at 100 x g for 5 min.</li>
	<li>Remove supernatant and resuspend pellet (soft) in 1 ml of MEM medium.</li>
	<li>Count cells using a haemocytometer and plate at 800,000 cells/coverslip in 500 &#181;l of MEM medium. Place in an incubator at 37 &#176;C with 6% CO2.</li>
	<li>Change the medium the following day (24-36 hr after the prep). Make a solution of MEM medium containing 10 &#181;M of the cell cycle inhibitor arabinofuranosyl cytidine (AraC).
	<ol>
		<li>For each coverslip, retain 250 &#181;l of the old medium in a fresh 24 well plate, aspirate the rest, add 250 &#181;l of normal MEM medium and aspirate this to wash the cells, then add the 250 &#181;l of old medium back to the cells and top up with 250 &#181;l of AraC-containing medium. 
		Note: Cells can be used from 6 days in vitro (DIV).</li>
	</ol>
	</li>
</ol>
4. Selectively Depleting the Microglia (Performed at 6 DIV)
<ol>
	<li>Add pure LME to 5 ml of a separate aliquot of MEM medium to give a final concentration of 150 mM LME.</li>
	<li>Return the solution to pH 7.4 and filter sterilize using a 28 mm polyether sulfone (PES) 0.2 &#181;m syringe filter.</li>
	<li>Dilute solution to a concentration of 50 mM (2 x LME) in MEM medium and place in a water bath at 37 oC for 10 min along with normal MEM medium for control cultures.</li>
	<li>Remove half (250 &#181;l) the MEM medium from CGC cultures and retain at 37 &#176;C.</li>
	<li>Treat cells with MEM medium containing 2 x LME (250 &#181;l), i.e. diluted 1:1 giving a well concentration of 25 mM, or with pre-warmed MEM medium without LME (250 &#181;l) for control cultures.</li>
	<li>Incubate cells at 37 &#176;C in a humidified atmosphere with 6% CO2 for 1 hr.</li>
	<li>Wash cells twice in fresh pre-warmed MEM medium to remove LME-containing media, and then replace the retained culture medium and an equal volume of fresh pre-warmed MEM medium to the corresponding wells.</li>
	<li>Return cultures to the incubator (humidified with 6% CO2 at 37 &#176;C) and leave to rest for 24 hr before any further treatment.</li>
</ol>

<ol>
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The authors declare that they have no competing financial interests.

The most important steps to ensure the successful selective elimination of microglia from CGC and/or mixed cultures are: 1) maintaining a sterile and healthy CGC culture; 2) filter sterilizing the LME-containing medium and returning the solution to pH 7.4; 3) keeping the retained CGC media and LME-containing media at 37 &#176;C to avoid heat shock; and 4) working quickly to reduce the time cells are kept outside the incubator.
We used 25 mM LME to deplete microglia from our CGC cultures &#8211; a concentration previously optimized in our laboratory. Nonetheless, some microglia still remain in culture following LME treatment, which some believe might reflect a population of differentiated, non-proliferating microglia. Indeed, Hamby et al. (2006) have found that 50-75 mM LME was required to eliminate microglia from high-density astrocyte monolayers13. We have also shown previously that in pure microglial cultures, 10 mM LME was sufficient to kill all the microglia, but only with a 24 hr incubation time14.
Alternative methods to rid cell culture models of contaminating microglia have recently come to light. Crocker et al. (2008) demonstrated a novel approach to eliminate microglia from astrocyte cultures by promoting neural stem cell (NSC) differentiation into astrocytes in postnatal (P0-P2) mice15. They found that this technique completely eliminated microglia from the cultures, although the mechanisms underlying the phenomena are unknown. In addition, Kumamaru et al. (2012) used liposomal clodronate &#8211; a biophosphonate known to induce apoptosis in macrophages &#8211; to eliminate microglia in primary astrocyte cultures from P3 mice16. This compound works in a similar fashion and at the same level of efficacy as LME; though the authors argue that as LME can be toxic to astrocytes under certain conditions due to its free diffusion into cells &#8211; disrupting astrocyte adhesion capacity13 &#8211; liposomal clodronate represents an improved method to deplete microglia from culture. We, however, found that 25 mM LME has no affect on astrocyte viability or proliferation.
The protocol described herein uses a specific concentration of LME to selectively eliminate microglia from neuron-enriched cultures of CGCs. This represents a powerful experimental approach to determine the influence of microglia on neurons under various conditions, and continues to provide important data to researchers investigating the role of neuroinflammation in neurological disorders.

The human brain comprises an estimated 85 billion neurons and a further 85 billion non-neuronal cells including glia1. For the greater part of the past 100 years neuroscientists have focused predominantly on the neuronal cell population, believing glial cells to be little more than passive support cells that provided structural support for the neurons &#8211; hence the Greek etymology of &#8216;glia&#8217; translated to English as &#8216;glue&#8217;. Recently, however, it has become increasingly evident that neuronal-glial interactions may be far more fundamental to basic aspects of neurobiology, neurophysiology, and the genesis and progression of many neurodegenerative diseases. Cerebellar granule cells (CGCs), the most abundant homogenous neuronal population in the human brain, dominate the cerebellum and make up more than 90% of its cellular constituents. Consequently, these cells have been used extensively in vitro as a model system for the study of neuronal development, function, and pathology2-6.
However, CGC cultures still contain microglia and other glia in arguably significant proportions. As a result, CGC data putatively displaying direct neuronal responses to different cell treatments may in fact arise &#8211; in part or in total &#8211; from the indirect secondary response of neighbouring glia in the culture. To assess this, we selectively eliminated microglial from CGC neuronal cultures with the aid of L-leucine methyl ester (LME). LME is a lysomotropic agent originally used to selectively destroy macrophages7, and has since been used to also selectively deplete microglia from neural, astrocyte, and mixed glial cultures8,9,10. LME is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Macrophages and microglia are characteristically rich in lysosomes, causing them to be particularly vulnerable upon exposure to LME treatment. This protocol provides a powerful, yet simple and easy way to ascertain the contribution of microglia in experiments utilizing CGC and other neuronal/glial culture systems.

<table><tbody><tr><td>Forceps&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F4142</td><td>The curved end facilitates removal of the cerebellum&amp;nbsp;</td></tr><tr><td>Micro-dissecting scissors</td><td>Sigma-Aldrich</td><td>S3146</td><td>Straight, sharp point facilitates rodent P4-7 dissection&amp;nbsp;</td></tr><tr><td>L-leucine methyl ester hydrochloride</td><td>Sigma-Aldrich</td><td>7517-19-3</td><td></td></tr><tr><td>EBSS solution&amp;nbsp;</td><td>Sigma-Aldrich</td><td>E7510-500 ml</td><td></td></tr><tr><td>Poly-D-lysine</td><td>Sigma-Aldrich</td><td>27964-99-4</td><td>Coat coverslips 1 day before use&amp;nbsp;</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>A9418</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich</td><td>P4417</td><td></td></tr><tr><td>DNase</td><td>Sigma-Aldrich</td><td>D5025</td><td></td></tr><tr><td>Soybean trypsin inhibitor&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T6414</td><td></td></tr><tr><td>Mouse anti-ED1 antibody&amp;nbsp;</td><td>Abcam</td><td>ab31630</td><td></td></tr></tbody></table>

selective depletion of microglia from cerebellar granule cell cultures using l-leucine methyl ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique &#8211; described here &#8211; is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer&#8217;s or Parkinson&#8217;s disease.

The ability of this technique to selectively eliminate microglia from CGC and/or mixed cultures relies upon the subsequent ability of the investigator to accurately identify and differentiate microglia from their surrounding cells. This can be achieved using a microglial-specific cell maker, such as isolectin-B4, as illustrated in Figure 1. As demonstrated in Figure 2, no observable changes to astrocyte and neuronal density and morphology were recorded with respect to each main treatment group. Importantly, no significant change in neuronal or astrocytic morphology were observed when cultures were treated with LME alone, supporting a microglial-specific action of the compound. Figure 3A shows no observable changes in astrocyte density and morphology from primary rodent astrocyte culture12, but does display a loss of contaminating microglia. In Figure 3B, as in A, no changes in astrocyte density and morphology were seen; however, a loss in contaminating microglial number and reactivity was observed. For a brief description of the immunocytochemistry (ICC) protocol used in this manuscript, please see reference11.
<img alt="Figure 1" src="/files/ftp_upload/52983/52983fig1.jpg" /> 
Figure 1: Characterization of LME treatment of CGCs. (A) Representative images from immunocytochemistry (ICC) experiments performed on CGC cultures after treatment with 25-75 mM LME for 1 hr, followed by wash-off. ICC was performed with DAPI (blue) for quantification of total cell number and those displaying apoptotic morphology, and isolectin-B4 (IB4) (green) for microglial identification and quantification. Scale bar = 30 &#181;m. (B) Quantification of cells displaying apoptotic morphology, defined as nuclear chromatin condensation (grey bars; % of total cell number in the field) and total microglial number, as a percentage of control levels (green bars). Unpaired Student&#8217;s t-tests were performed between the relevant controls and a specific treatment of LME; * p &#60;0.05, ** p &#60;0.01, *** p &#60;0.001; n = 3. (i.e. 3 independent repeats).
<img alt="Figure 2" src="/files/ftp_upload/52983/52983fig2.jpg" /> 
Figure 2: Characterization of the effects of 25 mM LME treatment on neurons and astrocytes in CGC cultures. Panel of representative images from ICC experiments performed on CGC cultures after treatment with 25 mM LME for 1 hr. ICC was performed with DAPI (blue), anti-&#946;-III-tubulin (green) for identification of changes in neuronal density and anti-GFAP (red) for identification of changes in astrocytic density and morphology. Negative control (-ve CTR) images represent CGC cultures wherein the primary antibodies were omitted. Scale bar = 30 &#181;m.
<img alt="Figure 3" src="/files/ftp_upload/52983/52983fig3.jpg" /> 
Figure 3: Further characterization of the effectiveness of 25 mM LME treatment in the removal of microglia from astrocytic cultures. (A) Panel of representative images from ICC experiments performed on primary astrocyte cultures after pre-treatment with 25 mM LME for 1 hr. ICC was performed with DAPI (blue), anti-GFAP (red) for identification of changes in astrocytic density and morphology, and anti-ED1 (green) for identification of microglial number. (B) Representative images from ICC experiments performed on primary astrocyte cultures after treatment with LPS (1 &#181;g/ml) for 24 hr following pre-treatment with 25 mM LME for 1 hr. ICC was performed as described in A but with anti-GFAP (green) and anti-iNOS (red) to identify activated microglia. Negative control (-ve CTR) images represent CGC cultures wherein the primary antibodies were omitted. Scale bar = 30 &#181;m.

Watch this Scientific Journal Video about Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester at JoVE.com

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...

selective-depletion-microglia-from-cerebellar-granule-cell-cultures

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.6K Views.  University of Washington. Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature. 

Video: Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

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 ...

Immunology and Infection

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

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

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

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

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

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 ...

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 ...

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.

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). ...

Modeling Reactive Oxygen Species-Induced Neuronal Death in Mouse Cerebellar Granule Neurons

Source: Laaper, M. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55871">Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.</a>&#160;J. Vis. Exp. (2017).
This video outlines the generation of a neuronal injury model using reactive oxygen species (ROS) in mouse primary cerebellar granule neurons. It details the process of inducing oxidative stress by adding high concentrations of hydrogen peroxide. ROS exposure causes DNA strand breaks and genomic instability. Additionally, the oxidation of cellular proteins impairs their functions, while lipid peroxidation compromises membrane integrity. This cascade leads to cytochrome c release and caspase activation, resulting in neuronal apoptosis.

1. Experimental Preparation
NOTE: The following stock solutions can be prepared and maintained until use.

	Dissection Solution
	
		Dissolve 3.62 g of ...

JoVE Encyclopedia of Experiments

Neuropathology

Neurodegenerative Diseases

Isolation of Microglia from the Cortex of an Adult Mouse Brain

Source: Zhou, L., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60347">Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing.</a> J. Vis. Exp. (2019)
The video demonstrates a technique for isolating microglia from the cortex of an adult mouse brain. First, the cortex undergoes segmentation and homogenization to remove neurons and other glial cells, preserving microglia. Following that, the cell suspension is exposed to anti-myelin magnetic microbeads and a magnetic field, effectively separating microglia and myeloid cells from myelin debris and cells containing myelin.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Mechanical Tissue Dissociation
...

Neuronal Culture Techniques

Primary Neuronal Culture

Isolation and Culture of Mouse Primary Cerebellar Granule Neurons

Source: Laaper, M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55871">Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.</a> J. Vis. Exp. (2017)
This video demonstrates the technique for isolating and culturing primary mouse cerebellar granule neurons. Cells are treated with an antimetabolite to eliminate proliferating glial cells and produce a pure culture of cerebellar granule neurons.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Experimental Preparation
NOTE: ...

Selectively Removing Microglia from a Rat Cerebellar Granule Neuron Culture

Source: Jebelli, J., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52983">Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester.</a> J. Vis. Exp. (2015)
This video demonstrates a technique for the selective removal of microglia from a primary rat neuronal culture. Cerebellar granule neurons are treated with an antimetabolite to eliminate proliferating glial cells. Additionally, they are treated with lysosome-targeting agents to deplete microglia from the culture.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Instruments, ...

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

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