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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Brain dissociation and magnetic microglial isolation
NOTE: All cell manipulations and resuspensions must be performed with a 1,000 &#181;L pipette with great caution. Applying a high mechanical action may activate or kill microglia cells.
<ol>
	<li>Transfer 12 brain pieces (~1.2 g) per dissociation tube containing dissociation mixture according to Table 1. For 24 pups, four C-Tubes will be needed (Figure 1A-B).</li>
	<li>Place C-Tubes on the dissociator (with the heating mode). Start the optimized NTDK program in the equipment according to the manufacturer&#39;s instructions (Figure 1D).</li>
	<li>Centrifuge at 300 x g for 20 s (at 4 &#176;C) to collect all the cells. Complete the mechanical dissociation by pipetting three times up and down with a 1,000 &#181;L pipette (Figure 1E).</li>
	<li>Transfer the cells to four 15 mL tubes + strainers. Rinse the strainers with 10 mL of 1x Hanks&#39; Balanced Salt Solution (HBSS) with Ca2+ and Mg2+ (HBSS+/+) (Figure 1F).</li>
	<li>Centrifuge at 300 x g for 10 min (at 4 &#176;C) and remove the supernatant. Carefully resuspend the pellet with 10 mL of HBSS+/+ (Figure 1G).</li>
	<li>Centrifuge at 300 x g for 10 min (at 4 &#176;C) and remove the supernatant. Carefully resuspend the pellet with 6 mL of sorting buffer (1x PBS + 0.5% BSA) (Figure 1H).</li>
	<li>Centrifuge at 300 x g for 10 min (at 4 &#176;C) and remove the supernatant. Add 200 &#181;L of CD11b-microbead solution per tube and resuspend carefully (Figure 1I).</li>
	<li>Incubate the tubes for 15-20 min at 4 &#176;C. Carefully resuspend the pellet with 6 mL of sorting buffer (Figure 1I-J).</li>
	<li>Centrifuge at 300 x g for 10 min (at 4 &#176;C) and remove the supernatant. Carefully resuspend the pellet with 8 mL of sorting buffer (Figure 1K).</li>
	<li>Follow the POSSEL program on the separator (see Table of Materials) to prepare eight columns. Transfer cells 1 mL by 1 mL on the column. Wait for all the cells to pass through before adding another mL. Elute CD11b+ cells on a sterile elution plate with 1 mL of sorting buffer (Figure 1L).</li>
	<li>Pool CD11b+ cells in a new 50 mL tube (Figure 1M).</li>
	<li>Centrifuge at 300 x g for 10 min (at 4 &#176;C) and remove the supernatant. Carefully resuspend the pellet with 10 mL of cold microglia medium (macrophage serum-free culture medium (SFM) + 1% of Penicillin-Streptomycin (P/S)) (Figure 1N).</li>
	<li>Count the CD11b+ cells. At P8, one should obtain ~650,000 cells per brain. 
	NOTE: In the present protocol, the cells were counted using an automated cell counter (see Table of Materials).</li>
	<li>Resuspend the cells in cold microglia medium to a final concentration of 650,000-700,000 cells/mL and dispense in cell culture plates. 
	NOTE: The 6-well plates are for Western Blot (2 mL per well); the 12-well plates are for RT-qPCR analysis (1 mL per well), and the 96-well plates are for phagocytic assay (250 &#181;L per well).</li>
</ol>
Table 1: Preparation of the dissociation mixture.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution</td>
			<td>For one C-Tube (&#956;L)</td>
			<td>For four C-tubes (&#956;L)</td>
		</tr>
		<tr>
			<td>Buffer X</td>
			<td>2850</td>
			<td>11400</td>
		</tr>
		<tr>
			<td>Enzyme P (Papain)</td>
			<td>75</td>
			<td>300</td>
		</tr>
		<tr>
			<td>Enzyme A (DNAse)</td>
			<td>15</td>
			<td>60</td>
		</tr>
		<tr>
			<td>Buffer Y</td>
			<td>30</td>
			<td>120</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>2970</td>
			<td>11880</td>
		</tr>
	</tbody>
</table>

<table><tbody><tr><td>Bovine Serum Albumin&amp;nbsp;</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-091-376</td><td></td></tr><tr><td>CD11b (Microglia) MicroBeads, h, m</td><td>Miltenyi Biotec</td><td>130-093-634</td><td></td></tr><tr><td>D-PBS (10x)</td><td>Thermo Scientific</td><td>14200067</td><td></td></tr><tr><td>Falcon Cell culture 12-well plate, flat bottom + lid</td><td>Dutscher&amp;nbsp;</td><td>353043</td><td></td></tr><tr><td>Falcon tubes 50 mL</td><td>Falcon tubes 50 mL</td><td>352098</td><td></td></tr><tr><td>Neural Tissue Dissociation Kit - Papain</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-092-628</td><td></td></tr><tr><td>Nucleocounter NC-200&amp;nbsp;</td><td>Chemometec</td><td></td><td></td></tr><tr><td>Nun EZFlip Top Conical Centrifuge Tubes</td><td>Thermo Scientific&amp;nbsp;</td><td>362694</td><td></td></tr><tr><td>OPTILUX Petri dish - 100 x 20 mm&amp;nbsp;</td><td>Dutscher&amp;nbsp;</td><td>353003</td><td></td></tr><tr><td>gentleMACS C Tubes (4 x 25 tubes)</td><td>Miltenyi Biotec</td><td>130-096-334</td><td></td></tr><tr><td>gentleMACS Octo Dissociator with Heaters</td><td>Miltenyi Biotec</td><td>130-096-427</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution (HBSS) +CaCl2 +MgCl2 10x</td><td>Thermo Scientific</td><td>14065049</td><td></td></tr><tr><td>Penicillin-streptomycin (10 000 U/mL)</td><td>Thermo Scientific&amp;nbsp;</td><td>15140122</td><td></td></tr><tr><td>Macrophage-SFM serum-free medium</td><td>Thermo Scientific</td><td>12065074</td><td></td></tr><tr><td>MACS BSA Stock Solution</td><td>Miltenyi Biotec</td><td>130-091-376</td><td></td></tr><tr><td>MACS SmartStrainers (70 &amp;mu;m), 4 x 25 pcs</td><td>Miltenyi Biotec</td><td>130-110-916</td><td></td></tr><tr><td>MultiMACS Cell24 Separator</td><td>Miltenyi Biotec</td><td></td><td></td></tr><tr><td>Multi-24 Column Blocks</td><td>Miltenyi Biotec</td><td>130-095-691</td><td></td></tr></tbody></table>

magnetic isolation of microglia from mouse pup brains

Source: Bokobza, C. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/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.

<img alt="Figure 1" src="/files/ftp_upload/21576/21576_Figure1.jpg" /> 
Figure 1: Schematic representation of brain dissociations and microglial cells&#39; isolation. (A-C) After P8 mouse brain dissection and removing olfactive bulbs and cerebellum, brains were first transferred to a Petri dish with HBSS+/+, and then to dissociation tubes containing the dissociation mixture. The C-tubes were placed on the dissociator (with the heating mode), and the NTDK program was started (<strong...

Magnetic Isolation of Microglia from Mouse Pup Brains

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 ...

Microglia &#8212; the brain-resident macrophages &#8212; express CD11b, a cell-surface integrin.
To isolate microglia from mouse pup brains, take cerebrum fragments in dissociation tubes containing buffer with papain, a proteolytic enzyme, and deoxyribonuclease.
Place the tubes on a mechanical dissociator. During the run, the dissociator mechanically disrupts the tissue.
Papain digests the tissue&#8217;s extracellular matrix, releasing cells, including microglia, into suspension. Deoxyribonuclease degrades free DNA in suspension, preventing inefficient cell isolation.
Centrifuge. Complete the mechanical dissociation by pipetting. Transfer the suspension through a cell strainer to remove debris and cell clumps, and obtain a homogeneous single-cell population.
Incubate with superparamagnetic microbeads functionalized with anti-CD11b antibodies. The microbeads specifically bind to CD11b on cells, including microglia.
Post-incubation, centrifuge. Remove the supernatant containing unbound beads. Resuspend the cells in buffer. Load onto a column placed on a magnetic separator. The column comprises a matrix with ferromagnetic beads.&#160;
When placed on the separator, the spheres within the column amplify the magnetic field, retaining the microbead-bound CD11b+ cells within the column, while other cells flow through.
Remove from the separator and use buffer to elute the CD11b+ cells from the column. Centrifuge the suspension and resuspend the CD11b+ cells in a microglia medium.
The isolated cells are ready for further analysis.

magnetic-isolation-of-microglia-from-mouse-pup-brains

Universidad de Cartagena UDEC

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Immunology

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Cell Isolation and Culture

208 Views. 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 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. 

Video: Magnetic Isolation of Microglia from Mouse Pup Brains - Concept

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

JoVE Journal

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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. ...

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 Microglia from Mouse Pup Brains

Transcript

Magnetic Isolation of Microglia from Mouse Pup Brains

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

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

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