isolation of microglia from the cortex of an adult mouse brain

Isolation of Microglia from the Cortex of an Adult Mouse Brain

First, use a razor blade to chop each brain region into fine pieces that are less than 1 cubic millimeter. Using a 1-milliliter pipette with the tip cut off, transfer the tissue pieces into pre-chilled Dounce homogenizers. Homogenize the tissue by slowly twisting the piston in and out of the Dounce homogenizer for 6 to 10 full strokes until no visible chunks are present. Then, transfer the dissociated tissues into 50-milliliter tubes through 70-micrometer strainers.
Rinse each Dounce homogenizer and piston with a total of 6 milliliters of cold medium A, then transfer the rinsing to a 15-milliliter tube for centrifugation. Centrifuge single-cell suspension at 400 times g and at 4 degrees Celsius for 5 minutes with a break at 5.
Rinse one large depletion column and three large selection columns in a magnetic separator with 3 milliliters of MCS. When the centrifugation for the tissue samples is complete, pipette out and discard the supernatant without disturbing the pellet. Resuspend the cells in MCS buffer that contains RNase inhibitor.
Add 100 microliters of myelin removal beads to each tube containing resuspended cells from the cortex and cerebellum, and add 50 microliters of myelin removal beads to each of the tubes containing resuspended cells from the hippocampus and striatum. Incubate the tubes on ice for 10 minutes.
After this, add MCS to the tube containing cortical cells to bring its volume up to 2 milliliters, and to the remaining tubes to bring each of their volumes up to 1 milliliter. Once the columns are empty of rinsing buffer, load 2 milliliters of cortical cells onto the large depletion column and 1 milliliter of each other cell suspension onto separate large selection columns. Next, wash the large depletion column once with 1 milliliter of MCS buffer, and wash each large selection column twice using 1 milliliter of MCS buffer for each wash.

isolation-of-microglia-from-the-cortex-of-an-adult-mouse-brain

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

EoE Sub Articles

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Mechanical Tissue Dissociation
NOTE: Keep cells and reagents cool all the time except during staining steps. This step should take ~30 min.
<ol>
	<li>Chop each brain tissue with a razor blade into &#60;1 mm3 fine pieces.</li>
	<li>Use 1 mL pipette (with tips cut off) to transfer tissue pieces into the pre-chilled Dounce homogenizers, each containing 2 mL of medium A with DNase and RNase inhibitor.</li>
	<li>Homogenize the tissue by slowly twisting the piston in and out of the Dounce homogenizer for 6&#8722;10 full strokes until no visible chunks are present. 
	NOTE: Douncing kills neurons and most other glial cells but leaves microglial cells rather intact. Both insufficient and over-douncing can lead to a low yield of cells.</li>
	<li>Transfer dissociated tissues into 50 mL tubes through 70 &#181;m strainers.</li>
	<li>Rinse each Dounce homogenizer and piston with a total of 6 mL of cold medium A and filter the rinsing solution through the same strainer into the corresponding tube.</li>
	<li>Transfer the single cell suspensions (about 8 mL each) into 15 mL conical tubes and centrifuge at 400 x g for 5 min at 4 &#176;C with brake = 5.</li>
</ol>
2. Myelin Removal
NOTE: This step should take ~60 min.
<ol>
	<li>Prepare 1 large depletion (LD) column (for the cortex) and 3 large selection (LS) columns (for the other 3 regions) in a magnetic separator (Table of Materials). Rinse each column with 3 mL of MCS buffer.</li>
	<li>Once centrifugation is finished, pipette and discard the supernatant without disturbing the pellet. For the cortex and cerebellum tissues, resuspend cells in 850 &#181;L of MCS buffer with 1.8 &#181;L of RNase inhibitor. For hippocampus and striatum tissues, resuspend cells in 400 &#181;L of MCS buffer with 0.9 &#181;L of RNase inhibitor. 
	NOTE: The columns (LD vs. LS) and the volume used for resuspension are optimized based on the amount of myelin present in the tissue. If other brain regions are assayed, these conditions may need to be adjusted depending on the size of the tissue and how much myelin may exist. The effectiveness of myelin removal can be estimated in step 2.9.</li>
	<li>Add 100 &#181;L of myelin removal beads each into cells from the cortex and cerebellum and add 50 &#181;L of myelin removal beads each into cells from the hippocampus and striatum.</li>
	<li>Gently mix the cells with beads and incubate the tubes on ice for 10 min.</li>
	<li>Bring the tube volume with cortical cells to 2 mL with MCS buffer and all others to 1 mL (i.e., add 1 mL for cortical cells; 500 &#181;L for hippocampal and striatal cells; no need to add buffer to cerebellar cells).</li>
	<li>Once columns are empty of rinsing buffer, place a 15 mL tube below each column. Load cortical cells (2 mL) onto the LD column and all others (1 mL each) onto the LS columns. Immediately use 1 mL of MCS buffer to wash the original tubes and load the solution onto the corresponding columns.</li>
	<li>Wash the LD column once with 1 mL of MCS buffer and wash each LS column twice with 1 mL of MCS buffer each wash. Continue to collect the flow-through solution during washing.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dumont #55 forceps</td>
			<td>Fine Science Tools</td>
			<td>11295-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce homogenizer, 2 ml</td>
			<td>Wheaton</td>
			<td>357422</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Worthington</td>
			<td>Cat# LS002007</td>
			<td>Working solution: 12500 units/ml</td>
		</tr>
		<tr>
			<td>Large depletion column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-901</td>
			<td></td>
		</tr>
		<tr>
			<td>Large selection column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MCS buffer</td>
			<td></td>
			<td></td>
			<td>Recipe: sterile-filtered 0.5% BSA, 2 mM EDTA in 1X PBS</td>
		</tr>
		<tr>
			<td>Medium A</td>
			<td></td>
			<td></td>
			<td>Recipe: 15 mM HEPES, 0.5% glucose in 1X HBSS without phenol red</td>
		</tr>
		<tr>
			<td>Myelin removal beads</td>
			<td>Miltenyl Biotec</td>
			<td>Cat# 130-096-433</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant RNase Inhibitor</td>
			<td>Takara Bio</td>
			<td>Cat# 2313B</td>
			<td></td>
		</tr>
		<tr>
			<td>Strainer (70 &#956;m)</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Round Bottom Polystyrene Tube, with Cell Strainer Cap</td>
			<td>Corning</td>
			<td>Cat# 352235</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10X), pH 7.4</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# 70011044</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M HEPES</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# 15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Hanks&#39; Balanced Salt Solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# 14185-052</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M EDTA, pH 8.0</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# 15575020</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Zhou, L., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/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
...

Take a freshly harvested mouse brain cortex.&#160;
Chop the tissue into small fragments. Transfer them into a chilled homogenizer containing a buffer with RNase inhibitors and DNases.&#160;
Homogenize the tissue optimally to release cells into suspension, selectively preserving microglia due to their ability to withstand shear forces and maintain membrane viability, while eliminating neurons and other glial cells.
Further, RNase inhibitors degrade RNases, while DNases degrade any contaminating DNA.
Pass the cell suspension through a cell strainer to remove tissue debris.
Transfer the cell suspension to a tube. Centrifuge and remove the supernatant containing enzymes. Resuspend the cells in a buffer.
Add anti-myelin magnetic microbeads. These microbeads target the myelin protein, binding myelin debris and oligodendrocytes.
Load the cell-bead mixture into a depletion column comprising a matrix with ferromagnetic spheres.&#160;
Under the applied magnetic field,&#160; the microbead-bound myelin debris and oligodendrocytes are retained within the column, while microglial cells pass through.
Wash the column with buffer and collect the flow-through containing microglia.

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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.

原代小胶质的快速和精细的CD11b电磁隔离带增强的纯度和多功能性

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. ...

科研

JoVE Journal

神经科学

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.

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

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 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.

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

通过磁活化细胞分选在脱髓鞘动物模型中分离小鼠原代小胶质细胞

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

Isolation of Microglia from the Cortex of an Adult Mouse Brain

成績單

Isolation of Microglia from the Cortex of an Adult Mouse Brain