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>

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<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>
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<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>
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</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 displayed20. In protocol step 2.8, care was taken to place brain tissue sections on an ice box for dissection and complete this step as soon as possible, thereby improving cell viability. In step 2.9, it is necessary to select appropriate tissue as one sample for both enzyme digestion and debris removal. Step 3.7 cannot be skipped when the effect of debris removal is not satisfactory. In step 4.3, the addition of PBS must be gentle to form different layers, ensuring that as much debris is removed as possible. All the steps in the protocol, except enzymatic digestion, should be performed on ice to ensure cell viability and reduce the transcriptional response of the microglia. Each step in the protocol is mild and retains high viability, so it is unnecessary to use the dead cells removal solution22,23.
The enzymatic dissociation method for mouse brain in the protocol has proven suitable for single-cell RNA-seq23. Although the enzymes in the protocol are mild, Marsh et al. have found that various enzymatic hydrolysis protocols at 37 &#176;C may significantly alter the transcriptome of microglia24. Setting a healthy control in the experiment for comparison can help to identify the differentially expressed genes of demyelination, thus eliminating the abnormal transcriptional response in the protocol. Meanwhile, toprevent this abnormal transcriptional state after enzymatic digestion, the protocol can be optimized by adding various transcription and translation inhibitors such as actinomycin D, triptolide and anisomycin at different steps. The use of these inhibitors can reveal the real gene expression of microglia in vivo without the setting of a control. Therefore, the addition of these inhibitors to the protocol can reduce the number of animals or specimens required in the experiment and is more economical for downstream analysis. However, it should be noted that some genes of microglia will be modulated by these inhibitors, which may limit the utilization of these inhibitors for future work24. Using the specialized dissociator instrument after the addition of enzyme mix 1 and enzyme mix 2 for tissue dissociation can also limit transcriptional responses in protocol step 323.
This protocol has some limitations. The purity of microglia is only approximately 85% in the protocol, which fails to comprise more than 90% of all single cells similar to other literatures19,22. The replacement of LS column with MS column, the absence of the filters, and the quality of microbeads may be the potential causes. If the purity of the isolated microglia fails to meet the requirements, prolonging the magnetic incubation time or passing the sorted cells through a second magnetic column will improve purity (Supplemental Figure S1). If the number of cells is low, tissue should be harvested from more mice and cell loss due to careless aspiration avoided. If the cell viability is low, replacing PBS in the loading buffer with RPMI 1640 medium could significantly improve the cell viability. It is also beneficial for cell viability to ensure that all the solutions used in the protocol are fresh, and the protocol is completed as soon as possible. It is important for users to balance the purity and the number of microglia obtained using this protocol.
The protocol utilizes the binding of microglia with CD11b beads and the enrichment in a magnetic field to purify the microglia. Therefore, purified microglia contain the contamination of myeloid cells, which is inevitable in this protocol. In contrast to FACS-the gold standard for sorting cells-the purity of microglia isolated by MACS is not sufficiently high for some elaborate applications such as deep sequencing, which limits its widespread usage. In addition, FACS for sorting microglia can eliminate the contamination of myeloid cells. However, MACS is a gentler method, retains more original morphological characteristics of glial cells especially astrocytes, takes less time for isolating microglia, and has a higher cell yield than FACS, suggesting that it can reduce the number of mice to be sacrificed and may be more suitable for time-sensitive experiments19,25. Meanwhile, MACS is more cost-effective and widely available and has lower technical requirements for experimenters. MACS has been proven the most reliable and consistent method to isolate microglia. Morphological analysis showed that microglia would not be activated by MACS, and RNA-seq has demonstrated that microglia isolated by this method maintain a resting state19,26.
Overall, the protocol is important for the survey of microglia in demyelinating diseases, and microglia isolated by this protocol can be utilized for various downstream applications such as quantitative RT-PCR and RNA-seq. The protocol utilizes neutral red dye to locate demyelinating lesions, which ensures the accuracy of lesion locations and reduces any confusion with normal tissue during sampling. Accurate localization of lesions can help focus on the effects of diseases and study the unique molecular phenotype and functional characteristics of microglia in demyelinating diseases. This will provide a basis for studying the mechanism of microglia in demyelinating diseases. This method of isolating microglia using magnetic CD11b beads can purify microglia with high cell viability and yield in a short time, with minimal requirements for experimental conditions, which allows widespread usage for studying microglia in different conditions.

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

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 purity of isolated microglia19,22. The fluorescence compensation was completed on the flow cytometer using unstained and single-stained control samples. Cells were gated by forward scatter-height (FSC-H) and side scatter-height (SSC-H); single cells were gated by forward scatter-area (FSC-A) and FSC-H. The proportions of microglia (identified as CD11b+CD45Intermediate) before and after the magnetic separation were tested by flow cytometry. The back-loop technique was used to adjust the gating strategy of flow cytometry analysis by determining the gate for the microglia. Compared with approximately 3 &#215; 104 cells and 6 &#215; 103 microglia before magnetic separation of each mouse brain (Figure 3A), MACS generally yields approximately 2.5 &#215; 103 cells and 2 &#215; 103 microglia per mouse brain (Figure 3B), which was approximately 85% of all single cells. However, nearly 3% of myeloid cells (identified as CD11b+CD45high) were present in the MACS-separated cells and were difficult to remove from the CD11b population in this protocol. The purity of the isolated microglia can be increased to 95% using two magnetic columns (Supplemental Figure S1). MACS sorting could capture approximately 33% of microglia in the brain mixture sample.
Microglia isolated using CD11b beads have high cell viability 
Fluorochrome 7-aminoactinomycin D (7-AAD) is often used to show the cell viability in the flow cytometry. 7-AAD+ cells represent dead cells19. The viability of MACS-separated microglia was approximately 95% by cell staining with 7-AAD (Figure 4).
Morphological analysis of microglia around demyelinating lesions sorted by MACS 
Morphological analysis showed that microglia were not activated by MACS. Microglia exhibit a typical longitudinal bipolar cell body after MACS, which is similar to microglial morphology after FACS19. Microglia around demyelinating lesions will be activated in the model of LPC-induced demyelination21. Microglia stained by ionized calcium binding adaptor molecule 1 (Iba-1) showed typical amebic morphology of activated microglia in this protocol. P2ry12 is a typical molecule of microglial resting state. There is a small fraction of branched and P2ry12+ microglia before MACS due to the expansion of the extent of the dissociated lesions. However, the existence of P2ry12+ microglia after MACS also indicates that MACS will not activate microglia (Supplemental Figure S2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63511/63511fig01.jpg" /> 
Figure 1: Images of the focal demyelinating lesions labeled by the neutral red dye. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63511/63511fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63511/63511fig02.jpg" /> 
Figure 2: Images of the three layers formed by centrifugation. The top two layers (layer 1 + layer 2) need to be removed in the debris removal process (see protocol step 4.5). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63511/63511fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63511/63511fig03.jpg" /> 
Figure 3: Flow cytometry analysis of microglia isolated from demyelination lesions of adult mice before and after MACS. (A) Schematic representation of gating strategy used in flow cytometry analysis before MACS: FSC-H and SSC-H for selecting cells, FSC-A and FSC-H for selecting single cells, and CD11b-FITC and CD45-APC for selecting myeloid cells (up) and microglia (down). (B) Schematic representation of the gating strategy used in flow cytometry analysis sample after MACS. The proportion of microglia has increased significantly after MACS: FSC-H and SSC-H for selecting cells, FSC-A and FSC-H for selecting single cells, and CD11b-FITC and CD45-APC for selecting myeloid cells (up) and microglia (down). Abbreviations: SSC-H = side scatter-height; FSC-H = forward scatter-height; FSC-A = forward scatter-area; CD45-APC = allophycocyanin-labeled CD45; CD11b-FITC = fluorescein isothiocyanate-labeled CD11b; MACS = magnetic-activated cell sorting. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63511/63511fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63511/63511fig04.jpg" /> 
Figure 4: FACS gating strategy for living/dead single cells after MACS. 7-AAD and SSC-H for selecting living cells after MACS. Abbreviations: SSC-H = side scatter-height; 7-AAD = 7-aminoactinomycin D; MACS = magnetic-activated cell sorting; FACS = fluorescence-activated cell sorting. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63511/63511fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure S1: Flow cytometry analysis of microglia isolated from adult demyelination mice. (A) Schematic representation of the gating strategy used in flow cytometry analysis sample before MACS. (B) Schematic representation of the gating strategy used in flow cytometry analysis sample after MACS using two magnetic columns. (C) Single-parameter histogram of CD11b-FITC and CD45-APC in flow cytometry after MACS using two magnetic columns. Abbreviations: SSC-H = side scatter-height; FSC-H = forward scatter-height; FSC-A = forward scatter-area; CD45-APC = allophycocyanin-labeled CD45; CD11b-FITC = fluorescein isothiocyanate-labeled CD11b; MACS = magnetic-activated cell sorting; CD45Int = intermediate CD45 expression. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63511/Figure S1.pdf" target="_blank">Please click here to download this File.</a>
Supplemental Figure S2: Immunofluorescence staining of microglia with Iba-1 and P2ry12 before and after MACS. (A) Images of microglia before MACS. (B) Images of isolated microglia after MACS. Scale bars = 20 &#181;m. Abbreviations: MACS = magnetic-activated cell sorting; Iba-1 = ionized calcium binding adaptor molecule 1; DAPI = 4&#39;,6-diamidino-2-phenylindole. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63511/Figure S2.pdf" target="_blank">Please click here to download this File.</a>

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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Universidad San Sebastian

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Neuroscience

3.5K Views.  Huazhong University of Science and Technology. Dit protocol helpt microglia te isoleren van de microdisseceerde demyelinisatielaesies van het volwassen muizenbrein om de microgliale kenmerken in vivo te bestuderen. Deze techniek bespaart tijd en stelt lagere eisen aan zowel experimentatoren als apparatuur. Deze methode helpt om microglia uit de hersenen van het dier te isoleren, vooral die microdisseceerde weefsels in verschillende ziektetoestanden.Begin met het microdisseceren van de laesies gelabeld door neutrale kleurstof rond h...

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

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.

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. ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

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

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

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

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

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

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

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

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. Microglia can adopt a large spectrum of activation phenotypes. Moreover, microglia that endorse pro-inflammatory phenotype is associated with both neurodevelopmental and neurodegenerative disorders. In vitro studies are widely used in research to evaluate potential therapeutic strategies in specific cell types. In this context studying microglial activation and neuroinflammation in vitro using primary microglial cultures is more relevant than microglial cell lines or stem-cell-derived microglia. However, the use of some primary cultures might suffer from a lack of reproducibility. This protocol proposes a reproducible and relevant method of magnetically isolating microglia from neonate pups. Microglial activation using several stimuli after 4 h and 24 h by mRNA expression quantification and a Cy3-bead phagocytic assay is demonstrated here. The current work is expected to provide an easily reproducible technique for isolating physiologically relevant microglia from juvenile developmental stages.

Primary microglia cultures are commonly used to evaluate new anti-inflammatory molecules. The present protocol describes a reproducible and relevant method to magnetically isolate microglia from neonate pups.

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. ...

Isolation of Hypothalamic Microglia from Mice Using MACS for In Vitro Assays

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

Microglia, as the resident macrophages of the brain, are essential for maintaining brain homeostasis. They shape neuronal circuits during development, survey their environment for debris or dead cells, as well as respond to infection and injury in the brain, among many other functions. However, their important role in neurodevelopment and synaptic plasticity and pathophysiology has not been fully defined, highlighting the need for further investigation. To gain a more comprehensive understanding of the role of microglia in these processes, we need to isolate microglia and characterize them genetically, metabolically, and functionally. However, the isolation of microglia from adult mice, especially from small brain structures, is challenging as they represent a small percentage of the total brain cells, and the yield of isolated microglia is often too low. Here, the magnetic isolation of microglia using CD11b+ microbeads allows us to sort microglial cells from the hypothalamus of a freshly perfused adult mouse brain. The current method allows us to achieve relatively high purity and yield in a short period while maintaining cell viability.

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 assays.

Microglia, as the resident macrophages of the brain, are essential for maintaining brain homeostasis. They shape neuronal circuits during development, ...

Isolation of Microglia from the Cortex of an Adult Mouse Brain

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

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

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Neuronal Culture Techniques

Primary Neuronal Culture

Magnetic Isolation of Microglia from Mouse Pup Brains

Source: Bokobza, C. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/62964">Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures.</a> J. Vis. Exp. (2022)
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.

Source: Bokobza, C. et al., Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures. J. Vis. Exp. (2022)
In this video, we ...

Immunology

Immune Systems and Components

Cell Isolation and Culture

Isolating Murine Primary Oligodendrocytes Using Immunomagnetic Beads

Source: Flores-Obando, R. E. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/57543">Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes.</a> J. Vis. Exp. (2018).
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.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Mouse ...

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

Source: Zhang, H. et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/63511">Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination.</a> J. Vis. Exp. (2022).
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.

 All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Materials

	&#160;Prepare ...

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

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