This work was supported by National Institutes of Health (NIH) Grants: NS088206 and ES026892. The W. Eugene and Linda Lloyd Endowed Chair to AGK and Deans Professorship to AK are also acknowledged.

Use of the animals and protocol procedures were approved and supervised by the Institutional Animal Care and Use Committee (IACUC) at Iowa State University (Ames, IA, USA)
1. Growing of Mixed Glial Cultures
<ol>
	<li>Decapitate 1- 2 day-old pups quickly with 5.5 inch operating scissors, and place the heads immediately in a 50 mL tube on ice. Note that this decapitation is the mode of euthanasia.</li>
	<li>In a laminar airflow hood, make a small incision in the skull and meninges using 4.5 inch straight micro-dissecting scissors. Begin cutting from the caudal end to the rostral end (nose). Get underneath the skin by using the opening formed by the decapitation.
	<ol>
		<li>After the incision, peel one of the hemispheres to the side. Then use a pair of curved or hooked tweezers to remove the entire brain.</li>
	</ol>
	</li>
	<li>Immerse the brain(s) in a new 50 mL tube containing 0.25% trypsin-ethylenediaminetetraacetate acid (EDTA) for 15 min in a 37 &#176;C water bath. Use 2 mL of trypsin-EDTA per brain.</li>
	<li>Wash the brain(s) with fresh Growth Media (10% FBS, DMEM/F12, 1% penicillin/streptomycin, 1% L-glutamine, 1% sodium pyruvate, and 1% non-essential amino acids) by adding and removing the media. Repeat this 4x.</li>
	<li>For each brain, plate two T-75 flasks containing growth media. Therefore, add 2 mL of growth media per brain to the tube. Thus, it will be an equivalent of 1 mL of homogenized brain with 8-9 mL of growth media per T-75 flask.</li>
	<li>Homogenize by triturating the brain(s) with pipettes of differing aperture sizes, in order from largest to smallest. When it is visible that the brain tissue is not getting smaller, transition to the next pipette. At the end of the trituration, the suspension should be clear, with no visible chunks.
	<ol>
		<li>Use a 25 mL pipette, 5 mL pipette, and then a 10 mL pipette sequentially.</li>
	</ol>
	</li>
	<li>Pass each homogenous brain suspension through a 70 &#181;m cell strainer to make it into a single cell culture.</li>
	<li>For each homogenized brain, plate two T-75 flasks containing growth media, as described in step 1.5 (1 mL of homogenized brain with 8-9 mL of growth media per T-75 flask).</li>
	<li>Change growth media after 6 d and grow until isolation on the 16th day.</li>
</ol>
2. Isolation of Microglial Cells
<ol>
	<li>After 16 days, remove the growth media from the flask and place it in a fresh 50 mL tube. Add 3 mL 0.25% trypsin-EDTA to each T-75 flask. Shake the flasks for 5 min at RT on an orbital shaker.
	<ol>
		<li>Centrifuge the removed growth media at 0.4 x g for 5 min and use to stop the trypsin-EDTA reaction in the subsequent step.</li>
	</ol>
	</li>
	<li>After shaking for 5 min, add a minimum of 4 mL of growth media (fresh or the used media from 2.1.1) to stop the trypsin-EDTA reaction.</li>
	<li>Triturate to ensure that all the cells have been detached.</li>
	<li>After trituration, pass the cells through a 70 &#956;m cell strainer to make it into a single cell culture, perform a cell count, and then spin down the cells at 0.4 x g for 5 min.</li>
	<li>For every 100 x 106&#160;cells (roughly 15 x T-75 flasks), use 1 mL of Recommended Media (2% FBS, DPBS (calcium&#160;and magnesium chloride-free), 1 mM EDTA) to resuspend the cell pellet. 
	NOTE: All following steps are tailored for a 1 mL separation.</li>
	<li>Take a 5 mL polystyrene tube, add 1 mL of Recommended Media, and mark the meniscus. Add Recommended Media up to 2.5 mL and mark this as well. Discard the Recommended Media and transfer the re-suspended cells to the 5 mL polystyrene tube.</li>
	<li>Add 50 &#181;L of rat serum for every 1 mL of suspended cells. Incubate for 5 min at RT.</li>
	<li>Prepare selection cocktail by mixing 25 &#181;L of component A and 25 &#181;L of component B. These components are proprietary.</li>
	<li>Add 50 &#181;L of the selection cocktail to the cells. Incubate for 5 min at RT. 
	NOTE: For purer cultures, repeat step 2.9 (recommended but not mandatory).</li>
	<li>Vortex microspheres for 45 s. Add 80 &#181;L of microspheres per 1 mL of sample. Incubate for 3 min at RT.</li>
	<li>Bring the volume to 2.5 mL in the polystyrene tube by adding the Recommended Media.</li>
	<li>Put the tube in the magnet for 3 min at room temperature. Adjust the incubation to increase purity. Slowly pour out the Recommended Media into a 15 mL tube with the magnet still in the polystyrene tube.</li>
	<li>Repeat step 2.12 three more times. Additional magnetic incubations may be performed to increase purity.</li>
	<li>Add 3 mL of growth media and count the number of cells using a cell counter.</li>
	<li>Plate cells accordingly in Poly-D-Lysine (PDL)-coated plates for treatments. Treat the cells 48 h after seeding inPDL-coated plates. This allows the cells to recover from the stress of separation. 
	NOTE: Check the purity of the culture using immunocytochemistry as described previously15.</li>
	<li>Plate the negative fraction (collected in a 15 mL tube), which mostly contains astrocytes, in T-75 flasks in the growth medium.</li>
	<li>After at least 6 h of incubation in 37 &#176;C incubator, change the medium and let it grow O/N. Split the astrocytes the next day for treatments.</li>
</ol>

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M., Cardona, A. 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L., Halliday, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+is+genetically+implicated+in+Parkinson's+disease.">Inflammation is genetically implicated in Parkinson's disease.</a> Neuroscience. 302, 89-102 (2015).</li><li>Xing, B., Bachstetter, A. D., Van Eldik, L. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M1+and+M2+immune+activation+in+Parkinson's+Disease:+Foe+and+ally?.">M1 and M2 immune activation in Parkinson's Disease: Foe and ally?.</a> Neuroscience. 302, 59-73 (2015).</li><li>Saijo, K., Glass, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+cell+origin+and+phenotypes+in+health+and+disease.">Microglial cell origin and phenotypes in health and disease.</a> Nat Rev Immunol. 11 (11), 775-787 (2011).</li><li>Paolicelli, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+pruning+by+microglia+is+necessary+for+normal+brain+development.">Synaptic pruning by microglia is necessary for normal brain development.</a> Science. 333 (6048), 1456-1458 (2011).</li><li>Floden, A. M., Combs, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+repetitively+isolated+from+in+vitro+mixed+glial+cultures+retain+their+initial+phenotype.">Microglia repetitively isolated from in vitro mixed glial cultures retain their initial phenotype.</a> J Neurosci Methods. 164 (2), 218-224 (2007).</li><li>Saura, J., Tusell, J. M., Serratosa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-yield+isolation+of+murine+microglia+by+mild+trypsinization.">High-yield isolation of murine microglia by mild trypsinization.</a> Glia. 44 (3), 183-189 (2003).</li><li>Gordon, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+magnetic+separation+method+for+high-yield+isolation+of+pure+primary+microglia.">A simple magnetic separation method for high-yield isolation of pure primary microglia.</a> J Neurosci Methods. 194 (2), 287-296 (2011).</li><li>Prinz, M., Priller, J., Sisodia, S. S., Ransohoff, R. 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S., Illes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenosine+receptor+signaling+in+the+brain+immune+system.">Adenosine receptor signaling in the brain immune system.</a> Trends Pharmacol Sci. 26 (10), 511-516 (2005).</li><li>Radulovic, M., Yoon, H., Wu, J., Mustafa, K., Scarisbrick, I. 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The authors declare that they have no competing financial interests.

Older microglial isolation methods have limited recoveries that are not appropriate for various protein analyses by Western blot and RNA analyses by qRT-PCR. The differential adherence and mild trypsinization methods are two common approaches with low microglial yields13,14,15. The column-based CD11b approach also has low recovery, but achieves greater purity than differential adherence and mild trypsinization1...

Microglia are Myb-independent resident macrophages of mesodermal origin, which differentiate from c-kit+/CD45- erythromyeloid progenitors in the blood islands of the yolk sac1,2. Once embryological microglia have colonized the central nervous system (CNS), they transition from an amoeboid to a ramified form3. These adult microglia are classified as surveillant since their dynamic ramifications probe the healthy brain parenchyma for potential insults4. Although microglia only contribute to approximately 10% of the CNS cell population, their ability to tile amongst each other ensures maximal scanning of the parenchyma4,5. Danger-associated molecular patterns (DAMPs), such as &#945;-synuclein6,7 and amyloid-&#946;8, or pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS)9, classically activate microglia to promote an inflammatory response characterized by reversion to the amoeboid active state and the production of nitric oxide, tumor necrosis factor-&#945; (TNF&#945;), interleukin 1&#946; (IL-1&#946;), IL-6, IL-12, and the chemokine C-C motif ligand 29,10,11. In neuroinflammatory conditions such as Parkinson&#39;s disease, in which pathogenic &#945;-synuclein has accumulated, a neurodegenerative cycle is created from the death of dopaminergic neurons, which release more aggregated &#945;-synuclein, further promoting classical activation of microglia7. Similar to peripheral macrophages, microglia may also have the ability to alternatively activate in the presence of the anti-inflammatory cytokines IL-4 and IL-10, giving them the potential of promoting neural repair and attenuating inflammation2,11. Aside from their immunological roles in the CNS, microglia have been described as vital regulators of neuronal circuitry by pruning synapses during development. For example, Cx3cr1-KO mice have less dense microglia and reduced synaptic pruning, which leads to an overabundance of dendritic spines, immature synapses, and the electrophysiological patterns of an underdeveloped CNS12. Understanding these physiological complexities and the diverse functional roles of microglia in the homeostasis of the CNS is critical to the search for therapeutics targeting neurodegenerative disorders.
In the area of neuroimmunology, in vitro experiments are highly desirable because of the greater feasibility for mechanistic studies, the lower maintenance costs, and for being less time- and labor-intensive. Furthermore, the ability to isolate cell populations is critical to delineate the functionality of those target cells under prescribed conditions. Numerous microglial isolation methods exist, but they are limited by their ability to obtain relatively high numbers and purity for broad experimentation13,14,15. For example, a cluster of differentiation 11b (CD11b) is a common surface marker of monocytes, macrophages, and microglia16. By exploiting CD11b, a method of magnetic separation was first described as a column-based approach that yielded ~99.5% purity and ~1.6 x 106 microglia per neonatal brain17. Our laboratory recently developed a column-free CD11b magnetic separation method15, which we performed in a polystyrene tube by tagging CD11b with a monoclonal antibody conjugated to phycoerythrin (PE). A bispecific secondary antibody to PE and dextran complexes with the PE. Once bound, dextran-coated magnetic particles are introduced, which bind to the dextran end of the antibody complex. Lastly, the polystyrene tube is placed in a magnet for microglial isolation. This approach doubled the yield to ~3.2 x 106 microglia per neonatal brain but at the cost of reducing purity to ~97%.
Herein, we demonstrate a rapid and refined column-free CD11b magnetic separation protocol (Figure 1). This improved method remains as feasible as our original column-free method since the price of the CD11b magnetic separation kit is the same. The completion time is reduced in half, which can be crucial to maximizing cell survival and yield. Notably, the purity achieved from this optimized method is ~&#62;99%, a marked improvement over the purity achieved from the original column-free method developed by our laboratory15. Most importantly, CD11b-PE is not utilized, eliminating the need to incubate away from light and allowing the use of the red channel for fluorescence microscopy. Lastly, as in the original CD11b method, an astrocytic fraction of high yield and purity is obtained with this improved method. Astrocytes are the most numerous glial cells in the CNS, leading to the idea that their homeostatic functions are indispensable in relation to pathophysiology18. These glial cells play a role in diverse physiological functions such as forming the blood-brain barrier, providing nutrient support, maintaining neurotransmitter homeostasis, forming glial scars in response to injury, neuroprotection, learning and memory, and neuroinflammation, exemplifying their investigatory potential in glial biology19. Morphology and functionality of microglia and astrocytes have been ascertained via confocal microscopy, Western blotting, quantitative Real-Time Polymerase Chain Reaction (qRT-PCR), Griess nitrite assay, and the Luminex multiplex cytokine assay. The refinement provided by this protocol offers increased confidence pertaining to microglial or astrocytic purity, broader application of fluorescence microscopy with the availability of the red channel, and saves time, all of which are important for in vitro experimentation.

<table>
	<tbody>
		<tr>
			<td>EasySep CD11b Separation Kit II</td>
			<td>StemCell Technologies</td>
			<td>18970</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>StemCell Technologies</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 (1:1) (1x)</td>
			<td>Life Technologies&#160;</td>
			<td>11330057</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Life Technologies&#160;</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-essential amino acids (100x)</td>
			<td>Life Technologies&#160;</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (100x)</td>
			<td>Life Technologies&#160;</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>13H469</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco by Life Technologies</td>
			<td>25200</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS</td>
			<td>Gibco by Life Technologies</td>
			<td>14190250</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Microglia isolated using CD11b Positive Selection kit II have high purity
Primary mouse microglia were isolated using the above mentioned protocol and plated on poly-D-lysine-coated coverslips to check the purity of isolation. Ten thousand cells were plated per well and immunocytochemical analysis was performed using ionized calcium-binding adaptor molecule 1 (Iba1) as a marker of microglia and glial fibrillary acidic protein (GFAP...

Watch this Scientific Journal Video about Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility at JoVE.com

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

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. ...

rapid-refined-cd11b-magnetic-isolation-primary-microglia-with

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

9.7K Views.  Iowa State University. 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.

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

The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33. To help improve this understanding, proton magnetic resonance spectroscopy (1H-MRS) can be used as it allows the in vivo quantification of brain metabolites such as &#947;-aminobutyric acid (GABA) and glutamate in a region-specific manner 41. In fact, a recent study demonstrated that 1H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31. Methodological factors to consider and possible modifications to the protocol are also discussed.

This article aims to describe a basic protocol for combining transcranial direct current stimulation (tDCS) with proton magnetic resonance spectroscopy (1H-MRS) measurements to investigate the effects of bilateral stimulation on primary motor cortex metabolism.

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of ...

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific frequency and in turn modulate ongoing cortical oscillatory activity. This neurotool allows the establishment of a causal link between endogenous oscillatory activity and behavior. Most of the tACS studies have shown online effects of tACS. However, little is known about the underlying action mechanisms of this technique because of the AC-induced artifacts on Electroencephalography (EEG) signals. Here we show a unique approach to investigate online physiological frequency-specific effects of tACS of the primary motor cortex (M1) by using single pulse Transcranial Magnetic Stimulation (TMS) to probe cortical excitability changes. In our setup, the TMS coil is placed over the tACS electrode while Motor Evoked Potentials (MEPs) are collected to test the effects of the ongoing M1-tACS. So far, this approach has mainly been used to study the visual and motor systems. However, the current tACS-TMS setup can pave the way for future investigations of cognitive functions. Therefore, we provide a step-by-step manual and video guidelines for the procedure.

Transcranial Alternating Current Stimulation (tACS) allows the modulation of cortical excitability in a frequency-specific fashion. Here we show a unique approach which combines online tACS with single pulse Transcranial Magnetic Stimulation (TMS) in order to "probe" cortical excitability by means of Motor Evoked Potentials.

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...

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 and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

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 ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

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 Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to use these cells for the purpose of studying the pathophysiology of amyotrophic lateral sclerosis (ALS) in the rat hSOD1G93A model. First, the protocol shows how to isolate and culture astrocytes and microglia from postnatal rat cortices, and then how to characterize and test these cultures for purity by immunocytochemistry using the glial fibrillary acidic protein (GFAP) marker of astrocytes and the ionized calcium-binding adaptor molecule 1 (Iba1) microglial marker. In the next stage, methods are described for dye-loading (calcium-sensitive Fluo 4-AM) of cultured cells and the recordings of Ca2+ changes in video imaging experiments on live cells.
The examples of video recordings consist of: (1) cases of Ca2+ imaging of cultured astrocytes acutely exposed to immunoglobulin G (IgG) isolated from ALS patients, showing a characteristic and specific response compared to the response to ATP as demonstrated in the same experiment. Examples also show a more pronounced transient rise in intracellular calcium concentration evoked by ALS IgG in hSOD1G93A astrocytes compared to non-transgenic controls; (2) Ca2+ imaging of cultured astrocytes during a depletion of calcium stores by thapsigargin (Thg), a non-competitive inhibitor of the endoplasmic reticulum Ca2+ ATPase, followed by store-operated calcium entry elicited by the addition of calcium in the recording solution, which demonstrates the difference between Ca2+ store operation in hSOD1G93A and in non-transgenic astrocytes; (3) Ca2+ imaging of the cultured microglia showing predominantly a lack of response to ALS IgG, whereas ATP application elicited a Ca2+ change. This paper also emphasizes possible caveats and cautions regarding critical cell density and purity of cultures, choosing the correct concentration of the Ca2+ dye and dye-loading techniques.

We present here a protocol on how to prepare primary cultures of glial cells, astrocytes, and microglia from rat cortices for time-lapse video imaging of intracellular Ca2+ for research on pathophysiology of amyotrophic lateral sclerosis in the hSOD1G93A rat model.

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to ...

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). ...