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 usin...

<ol>
	<li>Schulz, 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=A+lineage+of+myeloid+cells+independent+of+Myb+and+hematopoietic+stem+cells.">A lineage of myeloid cells independent of Myb and hematopoietic stem cells.</a> Science. 336 (6077), 86-90 (2012).</li><li>Prinz, M., Priller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+and+brain+macrophages+in+the+molecular+age:+from+origin+to+neuropsychiatric+disease.">Microglia and brain macrophages in the molecular age: from origin to neuropsychiatric disease.</a> Nat Rev Neurosci. 15 (5), 300-312 (2014).</li><li>Alliot, F., Godin, I., Pessac, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+derive+from+progenitors,+originating+from+the+yolk+sac,+and+which+proliferate+in+the+brain.">Microglia derive from progenitors, originating from the yolk sac, and which proliferate in the brain.</a> Brain Res Dev Brain Res. 117 (2), 145-152 (1999).</li><li>Ransohoff, R. M., Cardona, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myeloid+cells+of+the+central+nervous+system+parenchyma.">The myeloid cells of the central nervous system parenchyma.</a> Nature. 468 (7321), 253-262 (2010).</li><li>Kettenmann, H., Kirchhoff, F., Verkhratsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia:+new+roles+for+the+synaptic+stripper.">Microglia: new roles for the synaptic stripper.</a> Neuron. 77 (1), 10-18 (2013).</li><li>Su, X., 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=Synuclein+activates+microglia+in+a+model+of+Parkinson's+disease.">Synuclein activates microglia in a model of Parkinson's disease.</a> Neurobiol Aging. 29 (11), 1690-1701 (2008).</li><li>Dzamko, N., Geczy, C. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+p38alpha+MAPK+is+critical+for+LPS-induced+neuron+degeneration,+through+a+mechanism+involving+TNFalpha.">Microglial p38alpha MAPK is critical for LPS-induced neuron degeneration, through a mechanism involving TNFalpha.</a> Mol Neurodegener. 6, 84 (2011).</li><li>Panicker, N., 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=Fyn+Kinase+Regulates+Microglial+Neuroinflammatory+Responses+in+Cell+Culture+and+Animal+Models+of+Parkinson's+Disease.">Fyn Kinase Regulates Microglial Neuroinflammatory Responses in Cell Culture and Animal Models of Parkinson's Disease.</a> J Neurosci. 35 (27), 10058-10077 (2015).</li><li>Moehle, M. S., West, A. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+CNS+myeloid+cells+and+their+roles+in+neurodegeneration.">Heterogeneity of CNS myeloid cells and their roles in neurodegeneration.</a> Nat Neurosci. 14 (10), 1227-1235 (2011).</li><li>Marek, R., Caruso, M., Rostami, A., Grinspan, J. B., Das Sarma, ., J, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+cell+sorting:+a+fast+and+effective+method+of+concurrent+isolation+of+high+purity+viable+astrocytes+and+microglia+from+neonatal+mouse+brain+tissue.">Magnetic cell sorting: a fast and effective method of concurrent isolation of high purity viable astrocytes and microglia from neonatal mouse brain tissue.</a> J Neurosci Methods. 175 (1), 108-118 (2008).</li><li>Hasko, G., Pacher, P., Vizi, E. 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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+thrombin+receptor+modulates+inflammation+and+astrogliosis+to+improve+recovery+after+spinal+cord+injury.">Targeting the thrombin receptor modulates inflammation and astrogliosis to improve recovery after spinal cord injury.</a> Neurobiol Dis. 93, 226-242 (2016).</li><li>Ay, M., 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=Molecular+cloning,+epigenetic+regulation,+and+functional+characterization+of+Prkd1+gene+promoter+in+dopaminergic+cell+culture+models+of+Parkinson's+disease.">Molecular cloning, epigenetic regulation, and functional characterization of Prkd1 gene promoter in dopaminergic cell culture models of Parkinson's disease.</a> J Neurochem. 135 (2), 402-415 (2015).</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=Protein+kinase+Cdelta+upregulation+in+microglia+drives+neuroinflammatory+responses+and+dopaminergic+neurodegeneration+in+experimental+models+of+Parkinson's+disease.">Protein kinase Cdelta upregulation in microglia drives neuroinflammatory responses and dopaminergic neurodegeneration in experimental models of Parkinson's disease.</a> Neurobiol Dis. 93, 96-114 (2016).</li></ol>

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. ...

The overall goal of CD11b magnetic microglia isolation is to achieve a high purity end yield of postnatal microglia in a relatively short time period for the purposes of versatile in vitro experimentation. This method can help answer key questions in the field of neuroscience, such as the signaling mechanisms pertinent to neural inflammation. The main advantage of this technique over currently available methods is that the isolation takes approximately 30 minutes, with no sacrifices in purity, yield, or viability.Begin by transferring freshly decapitated heads from one-to two-day-old mouse pups to a laminar airflow hood. Then, use 4.5-inch straight micro-dissecting scissors to make a small incision in the skull and meninges by inserting the tips in the opening formed by the decapitation and cutting caudal to rostral. After making the incision, peel one of the hemisphere to the side.Then use a pair of curved or hooked tweezers to remove the entire brain. Transfer the brain to a 50-milliliter tube containing two milliliters of 0.25%trypsin EDTA, and incubate for 15 minutes in a 37-degree Celsius water bath. Wash the brain with fresh growth medium by adding and removing the medium.After completion of the washes, add two milliliters of growth medium per brain, and homogenize by triturating the brain. When the brain tissue does not get smaller, transition to a pipette with a smaller aperture. At the end of trituration, when the suspension is clear with no visible chunks, pass the suspension through a 70-micron cell strainer to create a single cell culture.Then pipette eight to nine milliliters of growth medium into T75 flasks, two flasks per brain, and add on milliliter of brain homogenate to each flask. Grow the cells until isolation on the sixteenth day. After sixteen days, transfer the growth medium from the flask to a fresh 50-milliliter tube.Add three milliliters of 0.25%trypsin EDTA to each T75 flask. Then shake the flasks for five minutes at room temperature on an orbital shaker. Centrifuge the growth medium at 0.4 times g for five minutes.After shaking for five minutes, add a minimum of four milliliters of the centrifuged culture medium to stop the trypsin EDTA reaction, and triturate to ensure that all the cells have been detached. Then pass the cells through a 70-micromolar cell strainer to create a single cell culture. Perform a cell count, and then spin down at 0.4 times g for five minutes.Next, take a five-milliliter polystyrene tube, add one milliliter of recommended medium, and mark the meniscus. Add recommended medium up to 2.5 milliliters, and mark this meniscus as well. Then, remove the medium, and re-suspend the pellet in 500 microliters of recommended medium.Transfer this mix into a five-milliliter polystyrene tube, and then dilute to the one milliliter mark. Then add 50 microliters of rat serum for every one milliliter of suspended cells, and incubate for five minutes at room temperature. During the incubation, prepare the selection cocktail by mixing 25 microliters of component A and 25 microliters of component B.Once the five minutes have elapsed, add 50 microliters of the selection cocktail to the cells, and incubate for a further five minutes at room temperature.Vortex the microspheres for 45 seconds. Then add 80 microliters of microspheres per one milliliter of sample, and incubate for three minutes at room temperature. Next, add recommended medium up to the 2.5 milliliter mark on the polystyrene tube.Put the tube in the magnetic holder for three minutes at room temperature. After the incubation, slowly pour out the medium into a 15-milliliter polystyrene tube still in the magnet. Repeat the magnetic separation three more times, each time adding recommended medium up to the 2.5 milliliter mark.After the last magnetic separation, add three milliliters of growth medium, and count the number of cells using a cell counter. Plate the cells in poly-D-lysine-coated plates for treatments. Plate the negative fraction from the 15-milliliter tube, which mostly contains astrocytes, in T75 flasks.After at least six hours of incubation in a 37-degree Celsius incubator, replace all the growth medium in the flasks containing the negative fraction before returning to the incubator. This image shows immunocytochemistry of an isolated microglial culture probed for IBA, a marker of microglia in the green channel, and GFAP, a marker of astrocytes, in the red channel. Hoechst staining reveals the presence of cell nuclei.The absence of GFAP-positive cells demonstrates that microglia isolated using the CD11b positive selection kit, too, have high purity. These immunoblotted proteins from an isolated microglial culture were probed for GFAP, which appears as an approximately 51-kilodalton band, and IBA1, which appears as an approximately 15-kilodalton band. Beta-actin was used as a loading control and appears at approximately 42-kilodaltons.A problem with the old isolation kit was that PE fluorescence could be observed in the red channel as seen here. The modified procedure does not produce any auto-fluorescence from the magnetic beads, as seen in the red channel here. The microglia isolated using this procedure can be used for signaling studies.Western blot analysis shows that Fyn and phospho-src tyrosine Y416 can be detected from microglia isolated with our newly-refined method. The negative fraction from the microglial separation contains astrocytes that can be used for signaling studies. Western blotting shows that the negative fraction contains GFAP-positive cells.Western blot analysis shows that Fyn and phospho-src tyrosine Y416 can be detected from the GFAP-positive cells. Following this procedure, other methods, like multi-channel fluorescence microscopy, western blotting, and quantitative PCR, can be performed to answer questions regarding gene expression and protein signaling. After watching this video, you should have a good understanding of how to utilize this rapid isolation technique for the purposes of microglial experimentation.

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Neuroscience

9.6K ビュー.  Iowa State University. The overall goal of CD11b magnetic microglia isolation is to achieve a high purity end yield of postnatal microglia in a relatively short time period for the purposes of versatile in vitro experimentation. This method can help answer key questions in the field of neuroscience, such as the signaling mechanisms pertinent to neural inflammation. The main advantage of this technique over currently available methods is that the isolation takes approximately 30 minutes, with no sacrifices in purity...

ビデオ: 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...

concomitant isolation of primary astrocytes and microglia for protozoa parasite infection

Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

The overall goal of this protocol is to instruct how to extract, maintain, and dissociate murine astrocyte and microglia cells from the central nervous system, followed by infection with protozoa...

obtaining a mixed glial cell culture from a neonatal mouse brain

The video demonstrated the method of isolating and culturing cells from a neonatal mouse brain. It involved enzymatic and mechanical dissociation of the brain, followed by culturing in growth media lacking neuronal growth factor to obtain a mixed glial...

Obtaining a Mixed Glial Cell Culture from a Neonatal Mouse Brain

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

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...

characterization and isolation of mouse primary microglia by density gradient centrifugation

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

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.

simultaneous isolation of principal central nervous system-resident cell types from adult autoimmune encephalomyelitis mice

Author Spotlight: Studying Neuroinflammatory Pathways Through Simultaneous Isolation of All Main CNS-Resident Cell Types 

To date, protocols for the simultaneous isolation of all principal central nervous system-resident cell types from the same mouse are an unmet demand. The protocol shows a procedure applicable in na&#239;ve and experimental autoimmune encephalomyelitis mice to investigate complex cellular networks during neuroinflammation and simultaneously reduce the required mice...

Simultaneous Isolation of Principal Central Nervous System-Resident Cell Types from Adult Autoimmune Encephalomyelitis Mice

magnetic isolation of microglia from mouse pup brains

In this video, we describe a protocol for isolating CD11b cells, including microglia, from mouse pup brains using magnetic-activated cell sorting. The brain tissues are homogenized to obtain single-cell suspensions, which are incubated with anti-CD11b antibody-coated microbeads, followed by a magnetic column-based separation to purify the CD11b+ cell...

Magnetic Isolation of Microglia from Mouse Pup Brains

Isolating All CNS Resident Cell Types Simultaneously from Autoimmune Mice

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rapid and efficient enrichment of mouse spinal cord microglia

Author Spotlight: Microglia Research on Spinal Cord Heterogeneity and Purification 

Microglia are regarded as some of the most versatile cells in the body, capable of morphological and functional adaptation. Their heterogeneity and multifunctionality enable the maintenance of brain homeostasis, while also being linked to various neurological pathologies. Here, a technique for purifying spinal cord microglia is...

Rapid and Efficient Enrichment of Mouse Spinal Cord Microglia

primary microglia isolation from postnatal mouse brains

Primary Microglia Isolation from Postnatal Mouse Brains

Primary cell culture is one of the primarily used approaches for studying microglial biology in vitro. Here, we developed a method for simple and rapid microglia isolation from the mouse postnatal day 1 (P1) to...

rapid and specific immunomagnetic isolation of mouse primary oligodendrocytes

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

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

isolation of cortical microglia with preserved immunophenotype and functionality from murine neonates

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

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...

isolation of hypothalamic microglia from freshly perfused adult mouse brain by magnetic-activated cell sorting  

Author Spotlight: Investigating the Impact of Nutrition on Mouse Brain Function and Metabolic Disorders 

We describe a protocol for isolating microglial cells from the mouse hypothalamus (or equivalent small brain structures) using magnetically activated cell sorting (MACS), in a relatively short time. The MACS-sorted hypothalamic microglia can be used for ex vivo analysis and can be plated to perform in vitro...

Isolation of Hypothalamic Microglia from Freshly Perfused Adult Mouse Brain by Magnetic-Activated Cell Sorting  

Transfer the cells to four 15-milliliter tubes with attached strainers. Rinse the strainers with 10 milliliters of HBSS with calcium and magnesium. Centrifuge for 10 minutes, and remove the supernatant with a 10-milliliter pipette. Carefully, add 10 milliliters of HBSS with calcium and magnesium, and resuspend the pellet. Again, centrifuge and remove the...

magnetic isolation of microglial cells from neonate mouse for primary cell cultures

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

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...

primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue

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

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...

co-culture of primary neurons and microglia for studying microglia-neuron interactions

Co-Culture of Primary Neurons and Microglia for Studying Microglia-Neuron Interactions

detection and isolation of apoptotic bodies to high purity

Detection and Isolation of Apoptotic Bodies to High Purity

A workflow using flow cytometry or differential centrifugation is developed to detect, quantify and isolate apoptotic bodies from an apoptotic sample to high purity.

brain isolation from neonatal mice for primary microglia culture

Brain Isolation from Neonatal Mice for Primary Microglia Culture

generating and co-culturing murine primary microglia and cortical neurons

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

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

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

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 ...

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 ...

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 ...

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 ...

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). ...

Extraction and Enzymatic Purification of Microglia from the Mouse Spinal Cord

The vertebral column defines a vertebrate and shapes the spinal canal, a cavity that encloses and safeguards the spinal cord. Proper development and function of the mammalian central nervous system rely significantly on the activity of resident macrophages known as microglia. Microglia display heterogeneity and multifunctionality, enabling distinct gene expression and behavior within the spinal cord and brain. Numerous studies have explored cerebral microglia function, detailing purification methods extensively. However, the purification of microglia from the spinal cord in mice lacks a comprehensive description. In contrast, the utilization of a highly purified collagenase, as opposed to an unrefined extract, lacks reporting within central nervous system tissues. In this study, the vertebral column and spinal cord were excised from 8-10 week-old C57BL/6 mice. Subsequent digestion employed a highly purified collagenase, and microglia purification utilized a density gradient. Cells underwent staining for flow cytometry, assessing viability and purity through CD11b and CD45 staining. Results yielded an average viability of 80% and a mean purity of 95%. In conclusion, manipulation of mouse microglia involved digestion with a highly purified collagenase, followed by a density gradient. This approach effectively produced substantial spinal cord microglia populations.

Microglia are regarded as some of the most versatile cells in the body, capable of morphological and functional adaptation. Their heterogeneity and multifunctionality enable the maintenance of brain homeostasis, while also being linked to various neurological pathologies. Here, a technique for purifying spinal cord microglia is described.

The vertebral column defines a vertebrate and shapes the spinal canal, a cavity that encloses and safeguards the spinal cord. Proper development and ...

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

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

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