AN is supported by the Institut Universitaire de France (IUF), the University of Bordeaux, the French Foundation for Brain Research (FRC), the GLN (Lipid-Nutrition Group), and the National Research Agency (ANR, PRC 2023-MicroNRJ). CA was supported by the Fondation pour la Recherche M&#233;dicale (FRM-ARF201809006962). This project has received funding from the European Union&#39;s Horizon Europe Research and Innovation Program under the MSCA Doctoral Networks 2021, No. 101072759 (FuElThEbRaiNIn healtThYaging and age-related diseases, ETERNITY.

All animal experiments described were conducted in strict compliance with the European Union recommendations (2013/63/EU) and were approved by the local ethical committee of the University of Bordeaux (CEEA50) and the French Ministry of Higher Education, Research, and Innovation (non-technical summary of approved project NTS-FR-619193 v.1, 23-12-2022).
The following protocol is performed on adult C57BL/6 mice with an average age of 2-4 months. However, it could be performed in all mice ages an...

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

<ol>
	<li>Ginhoux, F., Lim, S., Hoeffel, G., Low, D., Huber, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+differentiation+of+microglia.">Origin and differentiation of microglia.</a> Front Cell Neurosci. 7, 45 (2013).</li><li>Paolicelli, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+states+and+nomenclature:+A+field+at+its+crossroads.">Microglia states and nomenclature: A field at its crossroads.</a> Neuron. 110 (21), 3458-3483 (2022).</li><li>Ajami, B., Bennett, J. L., Krieger, C., Tetzlaff, W., Rossi, F. M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+self-renewal+can+sustain+CNS+microglia+maintenance+and+function+throughout+adult+life.">Local self-renewal can sustain CNS microglia maintenance and function throughout adult life.</a> Nat Neurosci. 10 (12), 1538-1543 (2007).</li><li>Nimmerjahn, A., Kirchhoff, F., Helmchen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resting+microglial+cells+are+highly+dynamic+surveillants+of+brain+parenchyma+in+vivo.">Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo.</a> Science. 308 (5726), 1314-1318 (2005).</li><li>Alexaki, V. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+isolation+from+adult+mouse+brain.">Microglia isolation from adult mouse brain.</a> Microglia: Methods Mol Biol. 1041, 17-23 (2013).</li><li>Schwarz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+fluorescence+activated+cell+sorting+to+examine+cell-type-specific+gene+expression+in+rat+brain+tissue.">Using fluorescence activated cell sorting to examine cell-type-specific gene expression in rat brain tissue.</a> J Vis Exp. (2015), (2015).</li><li>Lee, E., Eo, J. -. C., Lee, C., Yu, J. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+features+of+brain-resident+macrophages:+microglia+and+non-parenchymal+brain+macrophages.">Distinct features of brain-resident macrophages: microglia and non-parenchymal brain macrophages.</a> Mol Cells. 44 (5), 281-291 (2021).</li><li>Korin, 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=High-dimensional,+single-cell+characterization+of+the+brain's+immune+compartment.">High-dimensional, single-cell characterization of the brain's immune compartment.</a> Nat Neurosci. 20 (9), 1300-1309 (2017).</li><li>Mrdjen, D., 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=High-dimensional+single-cell+mapping+of+central+nervous+system+immune+cells+reveals+distinct+myeloid+subsets+in+health,+aging,+and+disease.">High-dimensional single-cell mapping of central nervous system immune cells reveals distinct myeloid subsets in health, aging, and disease.</a> Immunity. 48 (2), 380-395.e6 (2018).</li><li>Grabert, K., 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=Microglial+brain+region&#8722;dependent+diversity+and+selective+regional+sensitivities+to+aging.">Microglial brain region&#8722;dependent diversity and selective regional sensitivities to aging.</a> Nat Neurosci. 19 (3), 504-516 (2016).</li><li>Tan, Y. -. L., Yuan, Y., Tian, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+regional+heterogeneity+and+its+role+in+the+brain.">Microglial regional heterogeneity and its role in the brain.</a> Mol Psychiatry. 25 (2), 351-367 (2020).</li><li>Bustin, S. 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=The+MIQE+Guidelines:+minimum+information+for+publication+of+quantitative+real-time+PCR+experiments.">The MIQE Guidelines: minimum information for publication of quantitative real-time PCR experiments.</a> Clin Chem. 55 (4), 611-622 (2009).</li><li>. Available from: <a target="_blank" href="https://pamgene.com/wp-content/uploads/2022/03/1160-Preparation-Lysates-of-Cell-Lines-or-Purified-Cells-V4.2-2022-03.pdf">https://pamgene.com/wp-content/uploads/2022/03/1160-Preparation-Lysates-of-Cell-Lines-or-Purified-Cells-V4.2-2022-03.pdf</a> (2022)</li><li>Fonseca, M. I., 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=Cell-specific+deletion+of+C1qa+identifies+microglia+as+the+dominant+source+of+C1q+in+mouse+brain.">Cell-specific deletion of C1qa identifies microglia as the dominant source of C1q in mouse brain.</a> J Neuroinflamm. 14 (1), 48 (2017).</li><li>Simpson, D. S. A., Oliver, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROS+generation+in+microglia:+understanding+oxidative+stress+and+inflammation+in+neurodegenerative+disease.">ROS generation in microglia: understanding oxidative stress and inflammation in neurodegenerative disease.</a> Antioxidants. 9 (8), 743 (2020).</li><li>Galloway, D. A., Phillips, A. E. M., Owen, D. R. J., Moore, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+in+the+brain:+homeostasis+and+disease.">Phagocytosis in the brain: homeostasis and disease.</a> Front Immunol. 10, 790 (2019).</li><li>Marsh, S. E., 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=Dissection+of+artifactual+and+confounding+glial+signatures+by+single-cell+sequencing+of+mouse+and+human+brain.">Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain.</a> Nat Neurosci. 25 (3), 306-316 (2022).</li><li>DePaula-Silva, A. 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=Differential+transcriptional+profiles+identify+microglial-+and+macrophage-specific+gene+markers+expressed+during+virus-induced+neuroinflammation.">Differential transcriptional profiles identify microglial- and macrophage-specific gene markers expressed during virus-induced neuroinflammation.</a> J Neuroinflammation. 16 (1), 152 (2019).</li></ol>

The authors declare that they have no conflicts of interest.

The current protocol presents the isolation of hypothalamic microglia from freshly perfused adult mouse brain by Magnetic-Activated Cell Sorting. The results presented above confirm the purity and viability of isolated cells, as well as the efficacy of this method to functionally characterize microglia ex vivo, e.g. through phagocytic activity and ROS production quantification. Classical methods for the isolation of a specific cell population could also be used for microglial cells. Density gradient fractioning ...

Microglia correspond to 5-20% of the total neural cells and are the only glial cells that originate from erythromyeloid progenitors in the yolk sac and start to colonize the developing brain around embryonic day E9.51,2. They are long-lived cells with the ability to undergo self-renewal slowly, independently of bone-marrow-derived cells3. As some of the most highly dynamic cells, they are capable of acquiring diverse phenotypes in response to contextual and environmental cues2,4. Among the signals able to modulate their activity, central nervous system (CNS) damage, neuronal activity, as well as nutrients, are most potent. An increasing body of research has demonstrated the pivotal role of microglia in injury, neurodegenerative diseases, and obesity2,5,6. Nevertheless, the precise role of microglia in both physiological processes and pathophysiology requires further study. Therefore, their transcriptional, metabolic, and functional characterization under different conditions is of great importance and requires their isolation, since in situ studies are appropriate for DNA, RNA, and protein localization and qualitative comparisons, as well as morphological characterization of microglia.
There are various techniques described for microglia isolation, among which are the Percoll gradient method7, flow-cytometry cell sorting (FACS)8, and magnetically activated cell sorting (MACS). The selection of the appropriate method depends on the objectives of the study and the level of purity required for downstream applications. The isolation of pure microglia from the adult mouse brain, especially from small brain structures like the hypothalamus, is challenging due to the limited number of microglial cells. The automated gentle dissociation of the hypothalamus using Miltenyi technology allows the purification of a relatively high microglia yield in a short time with the ability to proceed up to eight samples at the same time with a gentleMACS Octo Dissociator.
Tissue homogenization is followed by microglia purification using the MACS column-based technology and the supermagnetic nano-sized CD11b+ beads. Therefore, all the samples are processed exactly in the same way yielding intact CD11b+ cells. It is noteworthy that CD11b is not exclusively present in microglia but is also expressed on other myeloid lineage cells, including macrophages and monocytes9. To limit this, brain perfusion with ice-cold saline solution prior to hypothalamus extraction (see protocol step 2.6) ensures the elimination of most myeloid cells, leaving only a potentially small fraction of resident macrophages or those adhered to blood vessels. The resident macrophages and monocytes of a healthy steady state CNS constitute a minor percentage of the total brain immune cells (~10% and &#60;2% respectively)10,11. Therefore, when brain cells are sorted with CD11b+ beads, both microglia and macrophages can be isolated, though the vast majority are microglia.
The end-yield of microglia and the maintenance of their viability let us perform ex vivo, as well as in vitro assays, with the advantage of analyzing specific brain regions of interest. The latest evidence has shown that the microglia population is highly heterogeneous, representing region-specific gene expression and morphological characteristics and function2,12,13. Therefore, the current protocol aims to isolate and analyze adult microglia in a region-specific manner. Indeed, we can characterize the genetic, transcriptional, and translational profiles of adult hypothalamic microglia using methods like RT-qPCR and RNA sequencing, as well as performing in vitro functional analysis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adult Brain Dissociation Kit</td>
			<td>Miltenyi</td>
			<td>130-107-677</td>
			<td>The kit contains buffer Z, buffer Y, enzymes A and P, Debris Removal Solution, buffer A (to dissolve enzyme A).</td>
		</tr>
		<tr>
			<td>Albumin Bovine FrV BSA</td>
			<td>EuroMedex</td>
			<td>04-100-812-C</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b (Microglia) MicroBeads, human and mouse - small size</td>
			<td>Miltenyi</td>
			<td>130-093-636</td>
			<td></td>
		</tr>
		<tr>
			<td>CellROX Green Flow Cytometry Assay Kit</td>
			<td>Invitrogen</td>
			<td>C10492</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tube, Snap-Pop Lid 15 mL</td>
			<td>CellTreat</td>
			<td>978449</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, calcium, magnesium, glucose, pyruvate</td>
			<td>Gibco</td>
			<td>14287-072</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS C Tubes</td>
			<td>Miltenyi</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS Octo Dissociator with Heaters</td>
			<td>Miltenyi</td>
			<td>130-096-427</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Phosphatase Inhibitor Cocktail (100x)</td>
			<td>Thermofisher</td>
			<td>78420</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Protease Inhibitor Cocktail, EDTA free (100x)</td>
			<td>Thermofisher</td>
			<td>78437</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (10x), calcium, magnesium, no phenol red</td>
			<td>Thermofisher</td>
			<td>14065056</td>
			<td></td>
		</tr>
		<tr>
			<td>Latex beads, carboxylate-modified polystyrene, fluorescent red</td>
			<td>Sigma-Aldrich</td>
			<td>L3280</td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler&#160;480 SYBR Green I Master</td>
			<td>Roche&#160;</td>
			<td>4707516001</td>
			<td>This reagent was used to perform PCR.&#160;</td>
		</tr>
		<tr>
			<td>MACS SmartStrainers (70 &#181;m)</td>
			<td>Miltenyi</td>
			<td>130-110-916</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro BCA Protein Assay Kit</td>
			<td>Thermofisher</td>
			<td>23235</td>
			<td></td>
		</tr>
		<tr>
			<td>M-PER Mammalian Extraction Buffer</td>
			<td>Thermofisher</td>
			<td>78503</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Columns</td>
			<td>Miltenyi</td>
			<td>130-042-201</td>
			<td>Referred as small columns in the protocol.&#160;</td>
		</tr>
		<tr>
			<td>MultiMACS Cell24 Separator Plus</td>
			<td>Miltenyi</td>
			<td>130-098-637</td>
			<td></td>
		</tr>
		<tr>
			<td>PBSS, pH 7.4&#160;</td>
			<td>Thermofisher</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>qScript XLT cDNA SuperMix</td>
			<td>Quanta biosciences</td>
			<td>733-1177</td>
			<td>The kit was used to syntesize cDNA.</td>
		</tr>
		<tr>
			<td>ReliaPrep RNA Miniprep Systems</td>
			<td>Promega</td>
			<td>Z6011</td>
			<td>The kit contains 1-Thioglycerol and BL buffer (referred as lysis buffer in the protocol) and it was used to isolate total RNA.</td>
		</tr>
		<tr>
			<td>Vacutainer safety-lok 21 G</td>
			<td>Becton Dickinson</td>
			<td>367282</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 

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

Our goal is to explore how nutrition affects brain function with an emphasis on the interaction between microglia and neurons. And we specifically focus on the hypothalamus, which is a key structure in the control of feeding behavior and energy balance. Most studies utilize genic mice to specifically target microglial processes and then combining this approach to histological or metabolic observations, but at the whole structural level, but we'll still have to achieve the cell level resolution.Isolating microglia from small brain structures is very challenging, and our protocol ensures a high yield and purity of valuable cells, which then allows for the study of the effect of the diet on microglial metabolism and function. Our protocol allows us to achieve relatively high purity and yield in a short period of time, and with lots of viable cells, even for small structures like the hypothalamus. Our research explores the role of microglia neuron communication in the resilience or vulnerability to develop metabolic conditions, and we know that individuals are extremely diverse in the way that respond to diet.And our data suggests that microglia could play a role in this diversity.

The evaluation of the end yield relies on the level of purity and the quantity of isolated cells and it could be determined by RT-qPCR, cell counting, and protein quantification. The purity of the isolated magnetic cells from the hypothalamus was confirmed by the gene expression analysis of CD11b, C1qa, Gad1, and GFAP with RT-qPCR (Figure 1). CD11b and C1qa are predominantly expressed by microglia in a steady state CNS16 and Gad1 and GFAP serve as a neuronal and astro...

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-hypothalamic-microglia-from-freshly-perfused-adult-mouse

Research

JoVE Journal

Neuroscience

Isolation of Microglial Cells from the Mouse Hypothalamus Using Magnetically Activated Cell Sorting for Flow Cytometry Analysis

To begin, place the previously isolated brain from the euthanized mouse on a Petri dish filled with ice. Position the brain upside down, such that the cortex is ventral, and the hypothalamus is visible dorsally on the superior surface. Then, dissect the hypothalamus using curved forceps.Collect the pieces of the hypothalamus in a dissociation or C-tube previously filled with 1.9 milliliters of buffer Z.Next, prepare 30 microliters of enzyme mix 2. In each C-tube, add 50 microliters of the enzyme contained in mix 1, and 30 microliters of enzyme mix 2. Place the C-tube containing hypothalamus pieces upside down in the dissociation with the heating brackets and run the adult brain dissociation kit program tube.In the meantime, place a 70 micron strainer on the top of a 15 milliliter snap tube. Wet the strainer with two milliliters of DPBS. After dissociation, empty the C-tube onto the strainer.Scratch the strainer with a 200 microliter tip to reduce cell loss by increasing filtration and reducing the adherence of homogenate on the filter. Wash the C-tube with five milliliters of DPBS and pour the suspension onto the strainer. Scratch the strainer with a 200 microliter tip.Centrifuge the tissue homogenate and aspirate the supernatant completely, before resuspending the pellet with an appropriate volume of cold DPBS. Immediately mix the cell suspension with 450 microliters of density gradient reagent, then, dropwise, gently overlay two milliliters of cold DPBS onto the cell suspension. Centrifuge the suspension with slow acceleration and slow break.From the three phases, carefully aspirate and discard the two top phases completely. Fill the tube with 1800 microliters of cold DPBS and gently invert three times. Now, centrifuge the mixture with full acceleration and full break.Aspirate the supernatant and resuspend the pellet in two milliliters of DPBS, containing 0.5%BSA. Centrifuge the cell suspension and resuspend the pellet in 90 microliters of DPBS, containing BSA. Add 10 microliters of the CD11b microbead mixture, and mix well with the pipette.Incubate the tube for 15 minutes at two to eight degrees Celsius in the refrigerator. Add one milliliter of DPBS, containing 0.5%BSA, and centrifuge the mixture. After aspirating the supernatant, resuspend the pellet in 500 microliters of DPBS, containing 0.5%BSA.Launch the parcel two program on the separator. Place the small columns in the magnetic field. Rinse the columns with 500 microliters of DPBS, containing 0.5%BSA.To collect the negative fraction of cells, place a collection plate under the columns and apply the cell suspension in the column. Wash the column with 500 microliters of DPBS, containing 0.5%BSA, and wait for the buffer to pass through the column. Transfer the total effluent of CD11b negative fraction from the wells of the collection plate to 15 milliliter tubes.Next, remove the column from the separator. Place the column on a five milliliter tube and pipette one milliliter of DPBS, containing BSA, into the column. Immediately, apply a plunger to flush out the magnetically labeled cell fraction.Count the CD11b positive cells on a hemocytometer, then centrifuge the cell suspension and discard the supernatant.

459 Views.  University of Bordeaux, INSERM, Neurocentre Magendie.  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 Mice Using MACS for In Vitro Assays

Summary