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 and conditions taking into consideration that the immune cell landscape of the CNS may be altered and the percentage of the resident macrophages and monocytes may be increased (e.g. in aging and neurodegeneration).
1. Preparation of solutions
NOTE: The following volumes and solutions are strictly referred to the isolation of Cd11b+ cells.
<ol>
	<li>Determine the total volume of D-PBS/0.5% Bovine Serum Albumin (BSA) required based on the number of samples. Prepare the solution using the commercially available D-PBS with Calcium and Magnesium and BSA powder (see the Table of Materials).</li>
	<li>Prepare labeled dissociation tubes (C-Tubes) and fill each with 1,900 &#181;L of buffer Z, provided in the adult brain tissue dissociation kit (see the Table of Materials). Thaw the enzymes and keep them on ice, together with all the buffers required in the following steps.</li>
	<li>Prepare and place the appropriate volume of 1x Phosphate-Buffered Saline (PBS) (see the Table of Materials) needed for the perfusion (step 2.6) on ice. Calculate 10 mL for each animal plus 10% in excess.</li>
	<li>For RT-qPCR analysis, prepare qPCR buffer per sample: 2.5 &#181;L of thioglycerol + 250 &#181;L of lysis buffer, using a commercially available kit to purify RNA (see the Table of Materials).</li>
	<li>For protein quantification, prepare lysis buffer by diluting the phosphatase inhibitor cocktail and protease inhibitor cocktail 1:100 in a mammalian extraction buffer (see the Table of Materials).</li>
	<li>For the detection of Reactive Oxygen Species (ROS) and phagocytosis assay, prepare 1x HBSS from the commercially available 10x HBSS (see the Table of Materials). Then, prepare FACS buffer by adding 0.2% BSA to 1x HBSS.</li>
</ol>
2. Removal of the brain
<ol>
	<li>Sedate the animal with a dose of 20 mg/kg of Xylazine; then, euthanize the mouse with an overdose of 400 mg/kg of pentobarbital and place it supine in a dissecting tray. 
	NOTE: Other anesthetic combinations to euthanize the animal can also work.</li>
	<li>Fill a 10 mL syringe with 10 mL of ice-cold 1x PBS and attach it to a butterfly 21 G needle.</li>
	<li>Once the mouse is completely anesthetized (verify by pinching the paws to ensure a lack of response), breathing stopped, and the heart is in fibrillation, tent the skin with forceps and open the thoracic cavity with scissors, at the level of the last ribs and the sternum.</li>
	<li>Use the scissors to make a deep cut along the sides of the ribcage towards the top of the ribcage to expose the heart.</li>
	<li>Insert the 21 G needle in the distal part of the left ventricle (ensure that approximately 1/3 of the needle is inside the ventricle). Immediately make an incision through the right atrium using the scissors.</li>
	<li>Infuse 10 mL of ice-cold 1x PBS to clean the brain from any circulating immune cells. 
	NOTE: Visually check the brain to ensure that it is clean and well-perfused.</li>
	<li>Cut the mouse head, carefully extract the brain, and place it on a Petri dish filled with ice and covered with aluminum foil. 
	NOTE: The Petri dish and foil help in seeing the brain pieces. It is beneficial to maintain a cool environment during the brain pieces chopping to limit metabolic activities.</li>
	<li>Isolate the hypothalamus using forceps and cut it into small pieces with razor blades.
	<ol>
		<li>To identify and dissect the hypothalamus, place the brain upside-down so that the cortex is ventral and the hypothalamus is visible dorsally on the superior surface. Separate the hypothalamus, which is located at the bottom of the brain on both sides of the third ventricle, using curved forceps. The average weight of the hypothalamus from adult C57BL/6 mice is 15 mg. 
		NOTE: The brain pieces must be as small as possible (&#60;0.5 mm). Cutting into small pieces is a critical step since it allows a better dissociation and a higher yield of cells.</li>
	</ol>
	</li>
	<li>Collect the pieces of the hypothalamus in dissociation tubes (C-Tubes) previously filled with 1,900 &#181;L of Buffer Z (step 1.2). 
	NOTE: To have a good yield of microglial cells that is enough to perform assays, pool a minimum of two (for protein quantification) or three (for qPCR and RNAseq) hypothalami per tube.</li>
</ol>
3. Tissue dissociation
NOTE: All the steps must be performed on ice. Do not vortex the cell suspension in any step; only mix carefully with a 1,000 &#181;L pipette.
<ol>
	<li>Prepare enzyme mix 2 according to Table 1 (calculating the total volume for all the tubes + 10%&#160;in excess).&#160;In each C-tube, add 50 &#181;L of the enzyme contained in mix 1 (see Table 1) and 30 &#181;L of enzyme mix 2. All the enzymes are provided in the adult brain tissue dissociation kit (see the Table of Materials).</li>
	<li>Close the C-tubes and place them upside-down in the dissociator with the heating brackets according to the manufacturer&#39;s instructions. Run the program 37C_ABDK_02.</li>
	<li>Prepare 15 mL snap tubes (see the Table of Materials) with 70 &#181;m strainers on top of them. Wet the strainer with 2 mL of D-PBS.</li>
	<li>When the dissociation/digestion program finishes, empty the C-Tube onto the strainer and scratch with a 200 &#181;L tip to reduce cell loss by increasing the cell filtration and reducing the adherence of tissue homogenate on the filter.</li>
	<li>Wash the tube with 2 x 5 mL of D-PBS, empty the washes onto the strainer, and scratch with a 200 &#181;L tip between the washes.</li>
	<li>Centrifuge at 300 &#215; g for 10 min at 4 &#176;C and aspirate the supernatant completely. Carefully resuspend the cell pellet with the appropriate volume of cold D-PBS according to Table 1.</li>
	<li>Immediately mix carefully with 450 &#181;L of density gradient reagent (Debris Removal Solution, see the Table of Materials) and overlay very gently with 2 mL of cold D-PBS (see Table 1). 
	NOTE: To obtain the different density gradient layers, the D-PBS must be overlaid gently drop by drop, with the least &#34;disturbance&#34; possible, 1 mL at a time with a 1 mL pipette.</li>
	<li>Centrifuge at 3,000 &#215; g for 10 min at 4 &#176;C with slow acceleration and slow brake (if 9 is equivalent to full, use 5).</li>
	<li>Three phases are formed. Aspirate the two top phases completely and discard them. Make sure to not aspirate the third bottom phase.</li>
	<li>Fill up with 1800&#160;&#181;L of cold D-PBS and gently invert the tube 3 times.</li>
	<li>Centrifuge at 1,000 &#215; g for 10 min at 4 &#176;C with full acceleration and full brake and aspirate the supernatant completely. Carefully resuspend the cell pellet by pipetting slowly up and down with 2 mL of D-PBS/BSA.</li>
</ol>
4. Magnetic isolation of CD11b+ cells
<ol>
	<li>Centrifuge at 300 &#215; g for 10 min at 4 &#176;C and aspirate the supernatant completely. Carefully resuspend the cell pellet in 90 &#181;L of buffer D-PBS/BSA.</li>
	<li>Add 10 &#181;L of the CD11b-microbead mixture and mix well with the pipette.</li>
	<li>Incubate for 15 min at 2-8&#160;&#176;C in the fridge, not on ice; wash the cells by adding 1 mL of D-PBS/BSA and centrifuge at 300 &#215; g for 10 min at 4 &#176;C; aspirate the supernatant completely. Resuspend carefully in 500 &#181;L of D-PBS/BSA.</li>
	<li>Launch the POSSEL2 program on the separator following the instructions on the screen. Place the small columns (see the Table of Materials) in the magnetic field. 
	NOTE: Either small or large columns can be chosen for magnetic cell sorting, according to the average number of cells expected. Small columns have a capacity of up to 1 &#215; 107 labeled cells and large columns up to 1 &#215; 108. When isolating cells from small structures, such as the hypothalamus, small columns are advised. The volumes of reagents are different when using large columns (see manufacturer&#39;s instructions).</li>
	<li>Wet the columns (see the Table of Materials) with 500 &#181;L of D-PBS/BSA. If the negative fraction of cells (CD11b-) is needed for further analysis, place a collection plate under the columns and then apply cell suspension in the columns.</li>
	<li>Perform three washing steps by adding 500 &#181;L of D-PBS/BSA at a time in the column, each time waiting for the buffer to pass through the column. 
	NOTE: it is important to prevent the upper part of the column from drying out, so it is advised not to wait until all the buffer has passed through.</li>
	<li>Transfer the total effluent of CD11b- fraction from the wells of the collection plate to 15-milliliter tubes.</li>
	<li>Remove the column from the separator, place it on a 5 mL tube, and pipette 1 mL of D-PBS/BSA into the column.</li>
	<li>Immediately flush out the magnetically labeled cell fraction by firmly applying the plunger supplied with the column.</li>
	<li>Eventually, count CD11b+ cells with a hemacytometer or any other method; expect ~15,000 cells per hypothalamus.</li>
	<li>Centrifuge the cell suspension at 300 &#215;g for 10 min at 4 &#176;C and discard the supernatant.</li>
</ol>
5. Analyses
<ol>
	<li>For RT-qPCR analysis, resuspend the cell pellets with qPCR buffer (for preparation, see step 1.3) and store the tubes at - 80 &#176;C until mRNA extraction. Isolate total RNA, then process and analyze it following the MIQE guidelines14. Finally, synthesize cDNA and perform qPCR using an RT-PCR device. See the Table of Materials for all the commercially available kits used.</li>
	<li>For protein quantification, wash the cell pellets with D-PBS (without BSA) at least 2x to eliminate BSA. Then, resuspend the cell pellets from two hypothalami with 20 &#181;L of lysis buffer. Proceed with protein quantification with a protein assay kit (see the Table of Materials). 
	NOTE: We followed the manufacturer&#39;s instructions15 to quantify proteins to perform the serine/threonine kinases activity assay.</li>
	<li>For the detection of Reactive Oxygen Species (ROS) in microglial cells, resuspend the pellet in 1x HBSS. Treat the cells following the manufacturer&#39;s protocol of the kit for Flow Cytometry Assay (see the Table of Materials). Then, resuspend treated cells in 200 &#181;L of FACS buffer (for preparation, see step 1.5) and perform FACS to detect oxidatively stressed cells.</li>
	<li>For phagocytosis assay, incubate the sorted cells in D-PBS/BSA with fluorescent latex beads (see the Table of Materials) for 30 min. Adapt the number of beads to have 4 beads per cell on average. Then centrifuge at 300 &#215; g for 7 min at 4 &#176;C, resuspend in 200 &#181;L of FACS buffer, and read the fluorescence emission by FACS (&#955;ex 575 nm; &#955;em 610 nm).</li>
</ol>

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

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	<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=single-cell+characterization+of+the+brain's+immune+compartment.">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 (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. -. 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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.

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...

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.

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, ...

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

Research

JoVE Journal

Neuroscience

766 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.

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

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

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 ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

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 ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.