Name: Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination (Video) | JoVE Journal
Uploaded: 2022-04-05T14:00:00.000+00:00
Duration: 4 min 53 s
Description: Learn magnetic-activated cell sorting to isolate primary microglia from demyelination lesions in adult mouse brains. This optimized protocol enables efficient study of microglial characteristics in vivo.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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

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

<ol><li>Ginhoux, F., et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGinhoux%2C+F+%22Science%22">Science</a>. 330 (6005), New York, N.Y. 841-845 (2010).</li>
<li>Schulz, C., et al. A lineage of myeloid cells independent of Myb and hematopoietic stem cells. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSchulz%2C+C+%22Science%22">Science</a>. 336 (6077), New York, N.Y. 86-90 (2012).</li>
<li>Paolicelli, R. C., et al. Synaptic pruning by microglia is necessary for normal brain development. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPaolicelli%2C+RC+%22Science%22">Science</a>. 333 (6048), New York, N.Y. 1456-1458 (2011).</li>
<li>Squarzoni, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+modulate+wiring+of+the+embryonic+forebrain%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">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/pubmed?term=((Layer+V+cortical+neurons+require+microglial+support+for+survival+during+postnatal+development%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">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., Martínez-Cerdeño, V., Noctor, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+regulate+the+number+of+neural+precursor+cells+in+the+developing+cerebral+cortex%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%3A+The+Official+Journal+of+the+Society+for+Neuroscience%22%5BJournal%5D)">Microglia regulate the number of neural precursor cells in the developing cerebral cortex.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 33 (10), 4216-4233 (2013).</li>
<li>Colonna, M., Butovsky, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+function+in+the+central+nervous+system+during+health+and+neurodegeneration%5BTitle%5D)+AND+%22Annual+Review+of+Immunology%22%5BJournal%5D)">Microglia function in the central nervous system during health and neurodegeneration.</a> Annual Review of Immunology. 35, 441-468 (2017).</li>
<li>Benmamar-Badel, A., Owens, T., Wlodarczyk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protective+microglial+subset+in+development%2C+aging%2C+and+disease%3A+Lessons+from+transcriptomic+studies%5BTitle%5D)+AND+%22Frontiers+in+Immunology%22%5BJournal%5D)">Protective microglial subset in development, aging, and disease: Lessons from transcriptomic studies.</a> Frontiers in Immunology. 11, 430(2020).</li>
<li>Yıldızhan, K., Nazıroğlu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+and+its+role+in+neurodegenerative+diseases%5BTitle%5D)+AND+%22Journal+of+Cellular+Neuroscience+and+Oxidative+Stress%22%5BJournal%5D)">Microglia and its role in neurodegenerative diseases.</a> Journal of Cellular Neuroscience and Oxidative Stress. 11 (2), 861-873 (2019).</li>
<li>Gutmann, D. H., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia%2Fbrain+macrophages+as+central+drivers+of+brain+tumor+pathobiology%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">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/pubmed?term=((Microglia+and+the+brain%3A+Complementary+partners+in+development+and+disease%5BTitle%5D)+AND+%22Annual+Review+of+Cell+and+Developmental+Biology%22%5BJournal%5D)">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/pubmed?term=((Microglial+dynamics+during+human+brain+development%5BTitle%5D)+AND+%22Frontiers+in+Immunology%22%5BJournal%5D)">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/pubmed?term=((Microglia+heterogeneity+in+the+single-cell+era%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">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/pubmed?term=((Neonatal+rat+primary+microglia%3A+isolation%2C+culturing%2C+and+selected+applications%5BTitle%5D)+AND+%22Current+Protocols+in+Toxicology%22%5BJournal%5D)">Neonatal rat primary microglia: isolation, culturing, and selected applications.</a> Current Protocols in Toxicology. , Chapter 12, Unit 12.17 (2010).</li>
<li>Yildizhan, K., Naziroglu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glutathione+depletion+and+parkinsonian+neurotoxin+MPP%28%2B%29-induced+TRPM2+channel+activation+play+central+roles+in+oxidative+cytotoxicity+and+inflammation+in+microglia%5BTitle%5D)+AND+%22Molecular+Neurobiology%22%5BJournal%5D)">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/pubmed?term=((Isolation+and+culture+of+microglia%5BTitle%5D)+AND+%22Current+Protocols+in+Immunology%22%5BJournal%5D)">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/pubmed?term=((New+tools+for+studying+microglia+in+the+mouse+and+human+CNS%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((A+simple+magnetic+separation+method+for+high-yield+isolation+of+pure+primary+microglia%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">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/pubmed?term=((Methodological+comparison+of+FACS+and+MACS+isolation+of+enriched+microglia+and+astrocytes+from+mouse+brain%5BTitle%5D)+AND+%22Journal+of+Immunological+Methods%22%5BJournal%5D)">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/pubmed?term=((Tracking+the+evolution+of+CNS+remyelinating+lesion+in+mice+with+neutral+red+dye%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((Alteration+of+synaptic+connectivity+of+oligodendrocyte+precursor+cells+following+demyelination%5BTitle%5D)+AND+%22Frontiers+in+Cell+Neuroscience%22%5BJournal%5D)">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/pubmed?term=((One+brain-all+cells%3A+A+comprehensive+protocol+to+isolate+all+principal+CNS-resident+cell+types+from+brain+and+spinal+cord+of+adult+healthy+and+EAE+mice%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">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/pubmed?term=((Dissociation+of+microdissected+mouse+brain+tissue+for+artifact+free+single-cell+RNA+sequencing%5BTitle%5D)+AND+%22STAR+Protocols%22%5BJournal%5D)">Dissociation of microdissected mouse brain tissue for artifact free single-cell RNA sequencing.</a> STAR Protocols. 2 (2), 100590(2021).</li>
<li>Marsh, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+cell+sequencing+reveals+glial+specific+responses+to+tissue+processing+%26+enzymatic+dissociation+in+mice+and+humans%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">Single cell sequencing reveals glial specific responses to tissue processing & enzymatic dissociation in mice and humans.</a> bioRxiv. , (2020).</li>
<li>Holt, L. M., Olsen, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+applications+of+magnetic+cell+sorting+to+analyze+cell-type+specific+gene+and+protein+expression+in+the+central+nervous+system%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Novel applications of magnetic cell sorting to analyze cell-type specific gene and protein expression in the central nervous system.</a> PLoS One. 11 (2), 0150290(2016).</li>
<li>He, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA+sequencing+analysis+reveals+quiescent+microglia+isolation+methods+from+postnatal+mouse+brains+and+limitations+of+BV2+cells%5BTitle%5D)+AND+%22Journal+of+Neuroinflammation%22%5BJournal%5D)">RNA sequencing analysis reveals quiescent microglia isolation methods from postnatal mouse brains and limitations of BV2 cells.</a> Journal of Neuroinflammation. 15 (1), 153(2018).</li>
</ol>

The authors have declared that no competing interests exist.

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

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

<table><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 &amp;micro;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

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's instructions. There are multiple literatures demonstrating that CD11b and CD45 antibodies are enough to check for the purity ...

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

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

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

Research

JoVE Journal

Neuroscience

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 population.

Magnetic Isolation of Microglia from Mouse Pup Brains

This video demonstrates a method to isolate primary oligodendrocyte cells from a neural cell suspension obtained from a mouse pup using magnetic separation. The technique employs magnetic beads coated with antibodies specific to oligodendrocyte antigens, enabling selective isolation of these cells.

Isolating Murine Primary Oligodendrocytes Using Immunomagnetic Beads

This video demonstrates a protocol for extracting microglial cells from demyelinating lesions in mouse brains using magnetic cell sorting. The protocol involves dissecting the lesioned brain tissue containing microglial cells, treating the tissue with specific enzymes to obtain a single-cell suspension, and utilizing microglial receptor-specific magnetic beads to isolate the microglial cells.

Isolating Microglia from Mouse Brain Demyelinating Lesions Using Magnetic Activated Cell Sorting

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.

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

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.

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

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

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

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

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

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.

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

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 assays.

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

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

Mouse Models of Periventricular Leukomalacia

Learn magnetic-activated cell sorting to isolate primary microglia from demyelination lesions in adult mouse brains. This optimized protocol enables efficient study of microglial characteristics in vivo.

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

Explore More Videos