Name: isolation of mouse primary microglia by magnetic-activated cell sorting in animal models of demyelination
Uploaded: 2022-04-05T14:00:00
Description: Watch this Scientific Journal Video about Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination at JoVE.com

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

All the animal procedures have been approved by the Institute of Animal Care Committee of Tongji Medical College, Huazhong University of Science and Technology, China.
1. Materials
<ol>
	<li>Prepare the following solutions before beginning the protocol.
	<ol>
		<li>Prepare the loading buffer by adding fetal bovine serum (FBS, 2%) to phosphate buffered saline (PBS).</li>
		<li>Add neutral red (NR) dye (final 1%) to PBS.</li>
	</ol>
	</li>
	<li>Prepare the following solutions ...

<ol>
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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>Squarzoni, P., 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=Microglia+modulate+wiring+of+the+embryonic+forebrain.">Microglia modulate wiring of the embryonic forebrain.</a> Cell Reports. 8 (5), 1271-1279 (2014).</li><li>Ueno, 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=Layer+V+cortical+neurons+require+microglial+support+for+survival+during+postnatal+development.">Layer V cortical neurons require microglial support for survival during postnatal development.</a> Nature Neuroscience. 16 (5), 543-551 (2013).</li><li>Cunningham, C. L., Mart&#237;nez-Cerde&#241;o, V., Noctor, S. 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H., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia/brain+macrophages+as+central+drivers+of+brain+tumor+pathobiology.">Microglia/brain macrophages as central drivers of brain tumor pathobiology.</a> Neuron. 104 (3), 442-449 (2019).</li><li>Hammond, T. R., Robinton, D., Stevens, 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+and+the+brain:+Complementary+partners+in+development+and+disease.">Microglia and the brain: Complementary partners in development and disease.</a> Annual Review of Cell and Developmental Biology. 34, 523-544 (2018).</li><li>Menassa, D. A., Gomez-Nicola, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+dynamics+during+human+brain+development.">Microglial dynamics during human brain development.</a> Frontiers in Immunology. 9, 1014 (2018).</li><li>Masuda, T., Sankowski, R., Staszewski, O., Prinz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+heterogeneity+in+the+single-cell+era.">Microglia heterogeneity in the single-cell era.</a> Cell Reports. 30 (5), 1271-1281 (2020).</li><li>Ni, M., Aschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+rat+primary+microglia:+isolation,+culturing,+and+selected+applications.">Neonatal rat primary microglia: isolation, culturing, and selected applications.</a> Current Protocols in Toxicology. , (2010).</li><li>Yildizhan, K., Naziroglu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione+depletion+and+parkinsonian+neurotoxin+MPP(+)-induced+TRPM2+channel+activation+play+central+roles+in+oxidative+cytotoxicity+and+inflammation+in+microglia.">Glutathione depletion and parkinsonian neurotoxin MPP(+)-induced TRPM2 channel activation play central roles in oxidative cytotoxicity and inflammation in microglia.</a> Molecular Neurobiology. 57 (8), 3508-3525 (2020).</li><li>Bohlen, C. J., Bennett, F. C., Bennett, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+microglia.">Isolation and culture of microglia.</a> Current Protocols in Immunology. 125 (1), 70 (2019).</li><li>Bennett, M. L., 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=New+tools+for+studying+microglia+in+the+mouse+and+human+CNS.">New tools for studying microglia in the mouse and human CNS.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (12), 1738-1746 (2016).</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> Journal of Neuroscience Methods. 194 (2), 287-296 (2011).</li><li>Pan, J., Wan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+comparison+of+FACS+and+MACS+isolation+of+enriched+microglia+and+astrocytes+from+mouse+brain.">Methodological comparison of FACS and MACS isolation of enriched microglia and astrocytes from mouse brain.</a> Journal of Immunological Methods. 486, 112834 (2020).</li><li>Baydyuk, 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=Tracking+the+evolution+of+CNS+remyelinating+lesion+in+mice+with+neutral+red+dye.">Tracking the evolution of CNS remyelinating lesion in mice with neutral red dye.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (28), 14290-14299 (2019).</li><li>Sahel, A., 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=Alteration+of+synaptic+connectivity+of+oligodendrocyte+precursor+cells+following+demyelination.">Alteration of synaptic connectivity of oligodendrocyte precursor cells following demyelination.</a> Frontiers in Cell Neuroscience. 9, 77 (2015).</li><li>Schroeter, C. B., 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=One+brain-all+cells:+A+comprehensive+protocol+to+isolate+all+principal+CNS-resident+cell+types+from+brain+and+spinal+cord+of+adult+healthy+and+EAE+mice.">One brain-all cells: A comprehensive protocol to isolate all principal CNS-resident cell types from brain and spinal cord of adult healthy and EAE mice.</a> Cells. 10 (3), 651 (2021).</li><li>Liu, L., 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=Dissociation+of+microdissected+mouse+brain+tissue+for+artifact+free+single-cell+RNA+sequencing.">Dissociation of microdissected mouse brain tissue for artifact free single-cell RNA sequencing.</a> STAR Protocols. 2 (2), 100590 (2021).</li><li>Marsh, S. 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The protocol proposes a method to isolate microglia around the demyelinating lesions, which can help study the functional characteristics of microglia in inflammatory demyelinating diseases. Microglia captured using CD11b beads exhibit high purity and viability. Critical steps in the protocol include the precise localization of foci and optimal microglial purification. In protocol step 2.1, it is necessary to inject the NR solution 2 h before sacrificing the mouse to ensure that the lesions can be accurately displayed<su...

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

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

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

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

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

Watch this Scientific Journal Video about Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination at JoVE.com

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

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

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

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