eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 
1. Preparation of the materials
<ol>
	<li>Magnetic-activated cell sorting (MACS) buffer: Prepare 2 mM ethylene diamine tetraacetic acid (EDTA) and 0.5% bovine serum albumin (BSA) (Table of Materials) in PBS-/-.</li>
	<li>Collagenase IV stock solution (20 U/&#956;L): Resuspend the powder in PBS+/+ (Table of Materials), aliquot, and store at -20 &#176;C. 
	NOTE: Collagenase IV requires MgCl2 to be fully active; do not freeze-thaw Collagenase IV solution.</li>
	<li>Anesthetic-analgesic solution: Mix 150 &#956;L of ketamine (150 mg/kg), 25 &#956;l of xylazine (5 mg/kg), and 330 &#956;l of buprecare (0.1 mg/kg) in 1 mL of PBS-/-. 
	NOTE: Prepare anesthetic-analgesic solution extemporaneously and do not keep it longer than a day.</li>
	<li>Heparin solution (100 U/mL): Resuspend the powder in PBS-/-.</li>
	<li>Prepare the infusion inset system connecting a tube to a decanter Erlenmeyer filled with PBS-/-. Connect a 23 G needle to the extremity of the infusion tube. Open the infusion inset and let the PBS run until there are no bubbles in the tube.</li>
	<li>Prepare the binocular loupe equipped with a light.</li>
</ol>
2. Housing of C57BL/6 mice
<ol>
	<li>For the analysis of CP (choroid plexuses) myeloid or T cells, use four mice for each and pool the four CP (two from each lateral ventricle, the third ventricle, and the fourth ventricle CP; for eight mice in total). Not pooling CP carries the risk of failing to detect the rare immune populations in CP due to their low abundance.</li>
	<li>Let the mice acclimatize for at least 7 days prior to any experimentation. Keep the mice under pathogen-free conditions at constant temperature and humidity, in a 12/12 h or 14/10 h light/dark cycle, with water and standard pellet food ad libitum.</li>
</ol>
3.&#160;&#8203;PBS perfusion and brain dissection
<ol>
	<li>Weight each mouse and inject 10 &#956;L/g of the anesthetic-analgesic mix solution (step 1.3) intraperitoneally.</li>
	<li>Wait around 30 min for efficient analgesia (check for the depth of anesthesia by pinching the mouse&#39;s digits).</li>
	<li>Position the anesthetized mouse flat on its back on the dissection support and tape its palms to the dissection support.</li>
	<li>Pinching the skin of the animal with forceps and using scissors, open the abdomen, the diaphragm, and the thorax to expose the heart. 
	NOTE: When the thorax is opened, the brain becomes anoxic. One must proceed precisely and swiftly through the next steps of the perfusion.</li>
	<li>Inject 20 &#956;L of 100 U/mL heparin solution directly into the left ventricle.</li>
	<li>With fine scissors, make an incision of at least 3 mm in the right atrium to allow the blood to flow out of the body.</li>
	<li>Immediately after, insert the 23 G needle placed at the extremity of the infusion tube through the tip of the left ventricle.</li>
	<li>Open the infusion system at the maximum and wait for complete perfusion: Using a 23 G needle at a flow rate of about 6 mL/min, the perfusion is complete in 3 min. 
	NOTE: The even discoloration of organs such as the liver attests to perfusion efficacy.</li>
	<li>Close the infusion system and remove the needle from the heart ventricle.</li>
	<li>Remove the tape from the mouse&#39;s palms. Place the mouse in a ventral position.</li>
	<li>Pinching the animal&#39;s skin with forceps and using scissors, remove the skin from the top of the head, from the eyes to the ears.</li>
	<li>With the help of scissors, cut the skull first between the eyes and then laterally from each eye to the spinal cord just above the masseter muscles. For this, use the extremity of scissors and proceed gently to avoid damaging the brain with the scissors.</li>
	<li>Open the skull with forceps by pinching it from the extremity between the eyes.</li>
	<li>Use scissors to cut the spinal cord and extract the brain with forceps, placing their two points on the lateral side of the brain to tilt it and place it in a Petri dish filled with ice-cold PBS+/+. The CPs are then immediately collected. 
	NOTE: Check for discoloration of the brain to verify perfusion efficacy.</li>
</ol>
4. Dissection of the Choroid Plexus from the brain
<ol>
	<li>Position the brain dorsal side up in the Petri dish and below the objectives of the binocular loupes.</li>
	<li>With the help of forceps to maintain the brain in place, insert the two ends of another forceps down through the midline between the hemispheres.</li>
	<li>Use the forceps to pull the cortex with the callosum and hippocampus away from the septum, exposing the lateral ventricle and a part of the third ventricle.</li>
	<li>Identify the lateral CP as a long veil lining the lateral ventricle that is flaring at both ends. Use the two ends of a thin forceps to catch the lateral CP. Be careful to collect the triangular posterior part that may be hidden by the posterior fold of the hippocampus.</li>
	<li>Pull the cortex, corpus callosum, and hippocampus of the contralateral hemisphere away from the septum to expose the entire third ventricle and the opposite lateral ventricle.</li>
	<li>Collect the third CP with fine forceps, which can be identified as a short structure with a granular surface aspect.</li>
	<li>Collect the other lateral CP.</li>
	<li>Insert the two ends of a forceps down between the cerebellum and midbrain. Detach the cerebellum from the pons and medulla to expose the fourth ventricle.</li>
	<li>Identify the fourth CP as a long globular structure with a granular surface aspect that lines the fourth ventricle from the lateral right end to the left end between the cerebellum and the medulla. Collect the fourth CP with fine forceps. 
	NOTE: Silane-base coating of forceps may prevent stickiness of CP on forceps.</li>
	<li>Repeat steps 3.1-4.9 for each of the other seven mice. In the meantime, the collected CP can be kept temporally in a tube placed on ice.</li>
</ol>
5. Preparation of samples for flow cytometry analysis
<ol>
	<li>Fill the tube containing all dissected CP to 750 &#956;L of PBS+/+. 
	NOTE: The deposition of dissected CP with thin forceps in the tube produces a small volume of PBS+/+. To avoid a possible loss of the CP tissue, it is better to complete the existing volume to 750 &#956;L than remove and replace it with fresh 750 &#956;L of PBS+/+.</li>
	<li>Add 15 &#956;L of 20 U/&#956;L Collagenase IV stock solution (see step 1.2) for a final concentration of 400 U/mL. 
	NOTE: DNAse I (150 &#956;g/mL) may prevent excessive cell clumping.</li>
	<li>Incubate CP with Collagenase IV at 37 &#176;C under mild agitation (300 RPM) for 45 min.</li>
	<li>Gently pipette up and down around 10 times to finalize the CP dissociation.</li>
	<li>Fill the CP tube to 1.5 mL with MACS buffer (see recipe step 1.1) to stop collagenase IV activity. 
	NOTE: Collagenase IV activity is stopped at low temperatures and inhibited by serum albumin.</li>
	<li>Centrifuge the cells at 500 x g, for 5 min, at 4 &#176;C. Discard the supernatant and wash the cells with 1.5 mL of MACS buffer.</li>
	<li>Centrifuge the cells at 500 x g for 5 min, at 4 &#176;C. Discard the supernatant and resuspend cells in 220 &#956;L of MACS buffer.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-mouse CD16/CD32</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>Albumin, bovine</td>
			<td>MP Biomedicals</td>
			<td>160069</td>
			<td>Blocking reagent</td>
		</tr>
		<tr>
			<td>APC anti-mouse CX3CR1</td>
			<td>BioLegend</td>
			<td>149008</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>APC anti-mouse TCRb</td>
			<td>BioLegend</td>
			<td>109212</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>APC-Cy7 anti-mouse CD4</td>
			<td>BioLegend</td>
			<td>100414</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>APC-Cy7 anti-mouse IA-IE</td>
			<td>BioLegend</td>
			<td>107628</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>BD FACSymphony A5 Cell Analyzer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometry analyzer</td>
		</tr>
		<tr>
			<td>BV711 anti-mouse Ly6C</td>
			<td>BioLegend</td>
			<td>128037</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Gibco</td>
			<td>17104-019</td>
			<td>Enzyme to dissociate CP tissue</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Scientific</td>
			<td>62248</td>
			<td>Live/dead marker</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td></td>
			<td></td>
			<td>Ion chelator</td>
		</tr>
		<tr>
			<td>fine scissors</td>
			<td>FST</td>
			<td>14058-11</td>
			<td>Dissection tool</td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD45</td>
			<td>BioLegend</td>
			<td>103108</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>Flow controller infusion inset</td>
			<td>CareFusion</td>
			<td>RG-3-C</td>
			<td>Blood perfusion inset</td>
		</tr>
		<tr>
			<td>FlowJo software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Analysis software</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>FST</td>
			<td>11018-12</td>
			<td>Dissection tool</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H3149-10KU</td>
			<td>Anticoagulant</td>
		</tr>
		<tr>
			<td>Imalgene</td>
			<td>Boehringer Ingelheim</td>
			<td></td>
			<td>Ketamine, anesthesic</td>
		</tr>
		<tr>
			<td>OneComp eBeads</td>
			<td>Invitrogen</td>
			<td>01-1111-42</td>
			<td>Control beads to realize compensation</td>
		</tr>
		<tr>
			<td>PBS-/-</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td>Buffer</td>
		</tr>
		<tr>
			<td>PBS+/+</td>
			<td>Gibco</td>
			<td>14040-091</td>
			<td>Buffer</td>
		</tr>
		<tr>
			<td>PE anti-mouse CD8a</td>
			<td>BioLegend</td>
			<td>100708</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>PE anti-mouse F4/80</td>
			<td>BioLegend</td>
			<td>123110</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>PE-Dazzle 594 anti-mouse CD11b</td>
			<td>BioLegend</td>
			<td>101256</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>Rompun</td>
			<td>Bayer</td>
			<td></td>
			<td>Xylazine, anesthesic</td>
		</tr>
		<tr>
			<td>thin forceps</td>
			<td>Dumoxel Biology</td>
			<td>11242-40</td>
			<td>Dissection tool</td>
		</tr>
		<tr>
			<td>Vetergesic</td>
			<td>Ceva</td>
			<td></td>
			<td>Buprenorphin, analgesic</td>
		</tr>
	</tbody>
</table>

isolating immune cells from mouse choroid plexuses

Source: Dominguez-Belloso, et al. <a href="https://app.jove.com/v/63487">Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses.</a> J. Vis. Exp. (2022)
This video demonstrates the procedure of isolating immune cells from mouse choroid plexuses. This procedure is used in neuroimmunology research to understand how immune cells in the choroid plexuses impact brain health and various neurological diseases.

Isolating Immune Cells from Mouse Choroid Plexuses

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 
1. ...

Position a mouse&#39;s brain dorsal side up.
Under a microscope, separate the cerebral hemispheres. Pull away the cortex and then the hippocampus to expose the lateral ventricles and the third ventricle.
Collect two lateral choroid plexuses. These are immune-cell-rich brain structures that line the lateral ventricles like a long veil.
Collect the third ventricle choroid plexus, which is a short structure with a granular surface.
Detach the cerebellum to expose the fourth ventricle.
Collect the fourth ventricle choroid plexus, which is a long globular structure with a granular surface.
Place all the choroid plexuses in a tube with buffered saline to maintain tissue integrity.
Add a digestive enzyme solution and incubate with agitation.
The enzyme breaks down the extracellular matrix and loosens the cells.
Add a suitable buffer to inactivate the enzyme.
Centrifuge to settle the cells. Discard the supernatant and resuspend the cells in a fresh buffer for further analysis.

isolating-immune-cells-from-mouse-choroid-plexuses

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JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

287 ビュー. 出典:Dominguez-Belloso, et al.微小解剖されたマウス脈絡叢からの免疫細胞の分離と特性評価。J. Vis. Exp.(2022年度) このビデオは、マウスの脈絡叢から免疫細胞を分離する手順を示しています。この手順は、脈絡叢の免疫細胞が脳の健康やさまざまな神経疾患にどのように影響するかを理解するための神経免疫学の研究で使用されます。 

ビデオ: マウス脈絡叢からの免疫細胞の単離 - 概念

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

Isolating Immune Cells from Mouse Choroid Plexuses

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Isolating Immune Cells from Mouse Choroid Plexuses