eoe sub articles

EoE Sub Articles

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Materials
<ol>
	<li>&#160;Prepare the following solutions before beginning the protocol:
	<ol>
		<li>Prepare the loading buffer by adding fetal bovine serum (FBS, 2%) to phosphate-buffered saline (PBS).</li>
		<li>Add neutral red (NR) dye (final 1%) to PBS.</li>
	</ol>
	</li>
	<li>Prepare the following solutions using the commercially available Adult Brain Dissociation Kit (see the Table of Materials).
	<ol>
		<li>To prepare enzyme mix 1, pipette 1.9 mL of Buffer Z and 50 &#956;L of Enzyme P into a 15 mL centrifuge tube.</li>
		<li>To prepare enzyme mix 2, pipette 20 &#956;L of Buffer Y and 10 &#956;L of Enzyme A into a 1.5 mL microcentrifuge tube.</li>
		<li>Prepare the debris removal solution.</li>
	</ol>
	</li>
</ol>
2. Mice perfusion and dissection
<ol>
	<li>Intraperitoneally (i.p.) inject the mouse with 500 &#956;L of 1% NR dye in PBS 2-3 h before anesthesia. 
	NOTE: Intraperitoneal injection of NR in demyelinating mouse models helps discern the lesions (Figure 1).</li>
	<li>Anesthetize the mouse with pentobarbital (50 mg/kg, i.p.), and ensure that the mouse is successfully anesthetized by examining the pain responses.</li>
	<li>Open the thoracic cavity of the mouse and expose the heart.</li>
	<li>Carefully cut off a small corner of the right atrium and intracardially perfuse the mouse with 20-30 mL of cold PBS from the left ventricle.</li>
	<li>Decapitate the mouse under anesthesia.</li>
	<li>Open the mouse&#39;s skull and carefully free the brain from the skull.</li>
	<li>Transfer the brain to the mouse brain slice mold and cut the brain into 0.1 cm slices.</li>
	<li>Remove the brain slices and place them in cold PBS in a Petri dish (60/15 mm). Using microsurgical forceps, microdissect the lesions labeled by NR dye around the corpus callosum under a stereomicroscope. 
	NOTE: Ensure the dissected tissue is as complete as possible to facilitate the subsequent transfer; the total dissection progress should be completed in 2 h.</li>
	<li>Transfer the dissected tissue to 15 mL centrifuge tubes containing the appropriate amount of cold loading buffer. 
	NOTE: Pool the corpus callosum tissue from 2-3 mice together in one tube as one sample.</li>
</ol>
3. Tissue dissociation
<ol>
	<li>Centrifuge the dissected tissue at 300 &#215;g for 30 s (after reaching this speed) to collect the sample at the bottom of the tube.</li>
	<li>Preheat enzyme mix 1 and 2 to 37 &#176;C in an incubator.</li>
	<li>Add 1,950 &#956;L of preheated enzyme mix 1 to one sample and digest in an incubator at 37 &#176;C for 5 min.</li>
	<li>Add 30 &#956;L of preheated enzyme mix 2 and mix gently.</li>
	<li>Digest in an incubator at 37 &#176;C for 15 min and mix gently every 5 min.</li>
	<li>Add 4 mL of cold PBS to the tube after digestion and shake gently.</li>
	<li>Place 70 &#956;m filters on 50 mL centrifuge tubes and prewet the filters with 500 &#956;L of cold PBS. Filter the dissociated tissue by passing the digested tissue samples through the filters, and transfer the filtered suspension in the 50 mL centrifuge tube to a 15 mL centrifuge tube. 
	NOTE: Skip this step if the amount of tissue is too small.</li>
	<li>Centrifuge the filtered tissue samples at 300 &#215;g for 10 min at 4 &#176;C and aspirate the supernatant slowly and completely. 
	NOTE: All the steps should be performed on ice except for the enzymatic digestion.</li>
</ol>
4. Debris removal
<ol>
	<li>Resuspend the cell pellet gently with 1,550 &#956;L of cold PBS.</li>
	<li>Add 450 &#956;L of cold solution for debris removal and mix well.</li>
	<li>Overlay the above mixture slowly and gently with 2 x 1 mL of cold PBS using a 1,000 &#956;L pipette. 
	NOTE: Tilt the centrifuge tube by 45 &#176; and slowly add PBS along the tube wall with the pipette.</li>
	<li>Transfer the tube slowly and gently to a centrifuge and spin at 4 &#176;C and 3,000 &#215; g for 10 min.</li>
	<li>Look for three layers after centrifugation. Aspirate the two top layers completely by using a 1,000 &#956;L pipette (Figure 2).</li>
	<li>Fill the tube up to 5 mL with cold loading buffer and gently invert the tube three times.</li>
	<li>Centrifuge at 4 &#176;C and 1,000 &#215; g for 10 min. Aspirate the supernatant completely and avoid disrupting the cell pellet.</li>
</ol>
5. Magnetic separation of microglial cells
<ol>
	<li>Resuspend the cell pellet with 90 &#956;L of loading buffer and add 10 &#956;L of CD11b (Microglia) beads.</li>
	<li>Mix well and incubate for 15 min at 4 &#176;C.</li>
	<li>Add 1 mL of loading buffer and gently pipette the liquid up and down with a 1,000 &#956;L pipette to wash the cells after incubating. Centrifuge the cells at 4 &#176;C and 300 &#215; g for 10 min and aspirate the supernatant completely to remove the unbound beads.</li>
	<li>Resuspend the cells in 500 &#956;L of loading buffer.</li>
	<li>Place the MS column with its separator for positive selection in the magnetic field.</li>
	<li>Rinse the column with 500 &#956;L of loading buffer to protect the cells and ensure the efficiency of magnetic sorting, following the manufacturer&#39;s protocol.</li>
	<li>Apply the cell suspension onto the MS column and discard the flowthrough containing unlabeled cells.</li>
	<li>Add 500 &#956;L of loading buffer to the column three times to wash away cells that adhere to the column wall and discard the flowthrough. 
	NOTE: Only add a new loading buffer for washing when the column reservoir is empty.</li>
	<li>Remove the column from the separator after washing the column and place it on a 15 mL centrifuge tube.</li>
	<li>Add 1 mL of loading buffer into the column and push the plunger to the bottom to flush out the magnetically labeled cells. Repeat this step three times to completely collect the magnetically labeled cells.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL Micro Centrifuge Tubes</td>
			<td>BIOFIL</td>
			<td>CFT001015</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Centrifuge Tubes</td>
			<td>BIOFIL</td>
			<td>CFT011150</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tubes</td>
			<td>BIOFIL</td>
			<td>CFT011500</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m Filter</td>
			<td>Miltenyi Biotec</td>
			<td>130-095-823</td>
			<td></td>
		</tr>
		<tr>
			<td>Adult Brain Dissociation Kit, mouse and rat</td>
			<td>Miltenyi Biotec</td>
			<td>130-107-677</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J Mice</td>
			<td>SJA Labs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b (Microglia) Beads, human and mouse</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-634</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>BOSTER</td>
			<td>PYG0001</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr>
			<td>MS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral Red</td>
			<td>Sigma-Aldrich</td>
			<td>1013690025</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>BOSTER</td>
			<td>PYG0021</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital</td>
			<td>Sigma-Aldrich</td>
			<td>P-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>MshOt</td>
			<td>MZ62</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating microglia from mouse brain demyelinating lesions using magnetic activated cell sorting

Source: Zhang, H. et al. <a href="https://app.jove.com/v/63511">Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination.</a> J. Vis. Exp. (2022).
This video demonstrates a protocol for extracting microglial cells from demyelinating lesions in mouse brains using magnetic cell sorting. The protocol involves dissecting the lesioned brain tissue containing microglial cells, treating the tissue with specific enzymes to obtain a single-cell suspension, and utilizing microglial receptor-specific magnetic beads to isolate the microglial cells.

<img alt="Figure 1" src="/files/ftp_upload/22606/22606_Figure1.jpg" /> 
Figure 1: Images of the focal demyelinating lesions labeled by the neutral red dye.
<img alt="Figure 2" src="/files/ftp_upload/22606/22606_Figure2.jpg" /> 
Figure 2: Images of the three layers formed by centrifugation. The top two layers (layer 1 + layer 2) must be removed in the debris removal process (see protoco...

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

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

	&#160;Prepare ...

Dissect stained demyelinating lesions from the corpus callosum of a mouse brain.
These lesions contain brain-resident microglia expressing the cell-surface integrin CD11b.&#160;
Collect the tissue and add a solution containing a proteolytic enzyme and DNase.
The enzyme degrades the extracellular matrix, while DNase degrades free DNA.
Add cold buffer and filter the suspension through a strainer to remove cellular clumps.
Centrifuge, discard the supernatant, and resuspend the pelleted cells in a density gradient medium.
Overlay with buffer and centrifuge to collect remaining debris in the upper layers; intact cells settle at the bottom.
Remove the debris and resuspend the cells in a buffer.
Add anti-CD11b magnetic beads to label the microglia.
Load the cells onto a column containing ferromagnetic spheres placed in the magnetic field of the magnetic separator.
Under the external magnetic field, the bead-bound microglia are retained in the column while unbound cells flow through.
Remove the column from the magnetic field. Add a buffer to elute the microglia.

isolating-microglia-from-mouse-brain-demyelinating-lesions-using

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Neuronal Culture Techniques

Primary Neuronal Culture

66 Views. Source: Zhang, H. et al. Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination. J. Vis. Exp. (2022). This video demonstrates a protocol for extracting microglial cells from demyelinating lesions in mouse brains using magnetic cell sorting. The protocol involves dissecting the lesioned brain tissue containing microglial cells, treating the tissue with specific enzymes to obtain a single-cell suspension, and utilizing microglial receptor-specifi...

Video: Isolating Microglia from Mouse Brain Demyelinating Lesions Using Magnetic Activated Cell Sorting - Concept

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 ...

JoVE Journal

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 ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

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

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

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

Transcript

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

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

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

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes