eoe sub articles

EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Sterile Poly-D-lysine (PDL) and Laminin Solutions
<ol>
	<li>At least one day prior to cell isolation, prepare poly-D-lysine (PDL) and laminin-coated plates. 
	NOTE: Both 6-well and 12-well plates were prepared depending on the experimental goals. Typically, 12-well plates were used for the quantitative analysis using western blots, whereas 6-well plates were used to hold autoclaved (sterile) coverslips for immunohistochemistry. 24-well plates were used to culture enteric glial cells (EGCs) for Ca2+&#160;flux imaging.</li>
	<li>Dilute stock solutions with ultrapure tissue culture grade, DNase-free, and RNase-free water in a tissue culture hood with HEPA-filtered air.</li>
	<li>Sterilizing glass coverslips
	<ol>
		<li>Hold the coverslip with sterile forceps and immerse coverslips in a beaker of 100% ethanol for 15 min. Allow the ethanol to evaporate in the tissue culture hood and then place it in the 6-well plates.</li>
		<li>Alternatively, place several coverslips individually on 2 mm thick blotting paper in a plastic container and then autoclave.</li>
	</ol>
	</li>
</ol>
2. Coating Plates
NOTE: Perform these steps under a sterile tissue culture laminar flow hood.
<ol>
	<li>Thaw 1 mg/mL PDL stock and dilute 1:10 with sterile, tissue culture-grade water so that the final concentration is 100 &#181;g/mL. Use 2 mL per well to coat 6-well plates, 1 mL to cover one well of a 12-well plate, and 0.5 mL for one well in the 24-well plates. Store the remaining diluted PDL in 12 mL aliquots at -20 &#176;C.</li>
	<li>After allowing the PDL to coat the wells for at least 1 h, remove the PDL with a sterile pipette. Allow the plates to air dry completely under a tissue culture laminar flow hood. 
	NOTE: After removing the PDL solution with a sterile pipette, it can be used twice more within 1 week after passing through a 0.22 &#181;m filter and storing at 4 &#176;C.</li>
	<li>Thaw the laminin stock on ice to avoid gel formation. 
	NOTE: Undiluted laminin becomes solid when warmed above 8 &#176;C.</li>
	<li>Dilute the laminin stock (0.5 mg/mL) 1:50 with sterile Dulbecco&#39;s Phosphate Buffered Saline (DPBS) to obtain a final concentration of 10 &#181;g/mL. Store the diluted laminin in 12 mL aliquots at -20 &#176;C.</li>
	<li>Add diluted laminin to the wells containing dried PDL. Use 1 mL to cover the 6-well surface and 0.5 mL to cover the bottom of the 12-well plates. For coverslips, add 400 &#181;L of diluted laminin to the coverslips to achieve surface coverage.</li>
	<li>Incubate the coated plates at 37 &#176;C for 2 h, if the plate is used the same day (quick coating). If not, seal the plates with plastic wrap and store them overnight at 4 &#176;C (slow coating). 
	NOTE: Sealing is critical to avoid drying and contamination.</li>
	<li>Remove the laminin solution carefully with a pipette to avoid scratching the surface. Gently wash the wells three times with sterile DPBS.</li>
	<li>Add complete glial growth media to each well and maintain the plates at 37 &#176;C until ready to add the EGC suspension. 
	NOTE: Poly-D lysine and laminin-coated plates can be stored for 3 days at 4 &#176;C. Coat the plates several days ahead and then store at 4 &#176;C; wash the plates with sterile Dulbecco&#39;s Phosphate Buffered Saline (DPBS) three times. Add 2 mL of sterile DPBS to each well after the final wash. Seal the plate with parafilm and store at 4 &#176;C. It is essential to ensure that the laminin does not dry out.</li>
</ol>
3. Preparation of Isolation Solutions
<ol>
	<li>Prepare the EDTA/HEPES/DPBS dissociation solution. For 500 mL of the solution, use sterile DPBS (without calcium and magnesium) to make a final solution of 10 mM HEPES and 5 mM EDTA. To 490 mL of the DPBS, add 5 mL of 1 M HEPES buffer and 5 mL of 0.5 M EDTA stock solution. Store at 4 &#176;C until use.</li>
	<li>Prepare the glial cell resuspension media using the reagents listed in&#160;Table 1.</li>
	<li>Prepare glial growth media using the reagents listed in&#160;Table 2. 
	NOTE: Glial growth media containing GDNF can be stored for one week at 4 &#176;C.</li>
</ol>
4. Removal of the Intestinal Segment
NOTE: Mice were allowed free access to food and water prior to euthanization by the isoflurane drop method and removal of a vital organ. C57BL/6 mice greater than 8 weeks of age were used. Both genders were used without noticeable differences in the preparation.
<ol>
	<li>Euthanize the mouse with an overdose of isoflurane and place it supine in a dissecting tray. Pin the extremities and sterilize the abdomen with 70% ethanol. Tent the skin with forceps and then open the abdomen with scissors.</li>
	<li>Lift the liver and identify the distal stomach/pylorus. Then, remove 7 cm of the proximal intestine. Be careful not to tear the intestine.</li>
	<li>Hold the pylorus with forceps while snipping away the mesentery.
	<ol>
		<li>Use blunt dissection with the back of the scissor to scrape away the adherent pancreas.</li>
		<li>Place the intestinal segment into ice-cold DPBS without Ca2+&#160;or Mg2+&#160;(Figure 1A). 
		NOTE: Other segments of the intestine or colon can also be removed using the same technique.</li>
	</ol>
	</li>
	<li>Use a 5- or 10-mL syringe attached to a blunt end 20 G needle to flush out the fecal contents with ice-cold DPBS (Figure 1B).</li>
	<li>Divide the intestine into 3 cm segments to remove the longitudinal muscle.&#160; NOTE: Intestinal segments from up to two mice are used per 30 mL of the HEPES/EDTA/DPBS isolation solution.</li>
	<li>Break off about 4 cm to separate the wooden stick from the cotton tip. Soak the wooden stick in DPBS (Figure 1C).</li>
	<li>Slip one of the intestinal segments smoothly onto the wetted stick (Figure 2A-C).</li>
	<li>Remove the adherent mesentery that is still attached using needle-nose forceps (Figure 2D).</li>
	<li>Use a clean razor blade or scalpel to make two longitudinal nicks along the intestine where the mesentery was attached (Figure 2E).
	<ol>
		<li>Wet the cotton tip with DPBS. Rub the wetted cotton tip longitudinally along the muscle to loosen the longitudinal muscle/myenteric plexus (LMMP). Then, move the cotton tip horizontally to tease away the LMMP from the circular muscle along the length of the intestinal segment during LMMP removal.</li>
		<li>Hold the tip of the stick between the thumb and forefinger while stabilizing the segment with the middle and fourth finger (Figure 2F). When complete, the LMMP will easily peel off the intestinal segment.</li>
	</ol>
	</li>
	<li>Discard the LMMP stripped from the intestinal tube and attached to the cotton swab unless harvesting EGCs from the myenteric plexus is desired.</li>
	<li>Use a razor blade to flay open the intestinal segment longitudinally to remove from the wooden stick.</li>
	<li>Store the stripped intestinal tissue (predominantly mucosa and submucosa plus circular muscle) in DPBS on ice. Repeat steps 4.4&#8211;4.11 until all intestinal segments are prepared.</li>
</ol>
5. Removal of the Epithelial Mucosa
<ol>
	<li>Cut the intestines into smaller pieces of ~0.5 cm with fine scissors.</li>
	<li>Place the tissue in a sterile 50 mL conical centrifuge tube containing 30 mL of ice-cold EDTA/HEPES/DPBS solution.</li>
	<li>Rock the tissue-containing solution at 4 &#176;C for 10 min at ~60 tilts per min.</li>
	<li>Pre-moisten a 5 mL plastic pipette by pipetting the EDTA/HEPES/DPBS buffer up and down one time, and then use the pre-moistened pipette to pipette the tissue and buffer suspension up and down (trituration) 20 times to dislodge the epithelial mucosa.</li>
	<li>Collect the tissue from the EDTA incubation by pouring the mixture through a 100 &#181;m nylon cell strainer. Discard the turbid mucous-filled flow-through (Figure 3).</li>
	<li>Place the tissue retained in the strainer into a new 50 mL conical centrifuge tube containing 30 mL of ice-cold EDTA/HEPES/DPBS using needle-nose forceps.</li>
	<li>Repeat the EDTA/HEPES/DPBS incubation a second time by rocking the tissue at 4 &#176;C for 10 min at ~60 tilts per minute to strip off additional epithelial mucosa.</li>
	<li>Pre-moisten a 5 mL pipette with the buffer and then pipette the tissue suspension up and down 20 times to dislodge the mucous cells. 
	NOTE: The tissue will tend to stick to the inside of the pipette as more of the epithelium is removed.</li>
	<li>Collect the tissue after the second incubation by pouring the mixture through a 100 &#181;m nylon cell strainer and discard the flow through. The second flow-through should be noticeably less turbid than the first (Figure 3).</li>
	<li>Repeat the 10 min EDTA incubations until the solution is nearly clear (about 3 to 4 incubations depending on the amount of tissue and the effectiveness of the trituration) (Figure 3).</li>
</ol>
6. Collection of Enteric Glial Cells from the Lamina Propria and Submucosa
<ol>
	<li>Transfer the tissue from the nylon strainer to a 15 mL conical centrifuge tube containing 5 mL of the commercially available cell recovery solution and rock for 25&#8211;30 min at 4 &#176;C.</li>
	<li>Triturate gently 10x to dissociate the enteric glial cells from the lamina propria.
	<ol>
		<li>Filter through a 40 &#181;m filter and collect the filtrate in a clean 50 mL tube.</li>
		<li>Rinse the tissue on the nylon filter with 1 mL of DPBS.</li>
		<li>Discard the tissue and transfer the ~5&#8211;6 mL of filtrate to a clean 15 mL conical centrifuge tube.</li>
		<li>Spin the filtrate at 2,000 x&#160;g&#160;in a swinging bucket centrifuge for 5 min at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Re-suspend the glial cell-containing pellet in at least 1 mL of resuspension buffer by gently pipetting the pellet up and down with a 500 &#181;L pipette tip. Avoid introducing bubbles. 
	NOTE: Adjust the resuspension volume as needed for the number of wells and plates. For example, 1.2 mL for one 6-well plate, 2.4 mL for two 6-well plates, 2.4 mL for one 12-well plate.</li>
	<li>Pipette 200 &#181;L of the cell suspension into each well of the 6-well or 100 &#181;L for a 12-well PDL-laminin coated plates containing glial growth media.</li>
	<li>Do not disturb the dishes after adding the cells to the plates and placing in the 37 &#176;C incubator to allow the maximum number of cells to adhere. 
	NOTE: A healthy preparation of cells will attach within 6&#8211;8 h, but wait for at least 24 h before changing the media by gently pipetting off the media along with non-adherent cells.</li>
	<li>Use the cells for experiments 3&#8211;4 days after they are placed into culture (Figure 4).</li>
</ol>
Table 1. Composition of Glial Cell Resuspension Media.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Components&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; </td>
			<td>Volume to be added</td>
			<td>Final Concentration</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>44.5 mL</td>
			<td>_</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>5 mL</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>500 &#181;L</td>
			<td>100 IU/mL Pen</td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td></td>
			<td>100 &#181;g/mL Strep</td>
		</tr>
		<tr>
			<td>Gentamicin (50 mg/mL stock)</td>
			<td>20 &#181;L</td>
			<td>20 &#181;g/mL</td>
		</tr>
	</tbody>
</table>
Table 2. Composition of Glial Cell Growth Media
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Components&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; </td>
			<td>Volume to be added</td>
			<td>Final Concentration</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>44 mL</td>
			<td>_</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>5 mL</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (100x)</td>
			<td>500&#181;L</td>
			<td>100 IU/mL (Pen)</td>
		</tr>
		<tr>
			<td>100 &#181;g/mL (Strep)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin (50 mg/mL stock)</td>
			<td>20 &#181;L</td>
			<td>20 &#181;g/mL</td>
		</tr>
		<tr>
			<td>GDNF (10 &#181;g/mL stock)</td>
			<td>50 &#181;L</td>
			<td>10 ng/mL</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Poly-D lysine (1 mg/ml stock)</td>
			<td>Sigma</td>
			<td>A-003-E</td>
			<td>Dilute 1:10</td>
		</tr>
		<tr>
			<td>Laminin (0.5 mg/ml stock)</td>
			<td>Sigma</td>
			<td>L4544</td>
			<td>Dilute to 10 &#181;g/mL on ICE</td>
		</tr>
		<tr>
			<td>EDTA (0.5M)</td>
			<td>Lonza</td>
			<td>51201</td>
			<td>Dilute 1:100 in DPBS</td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Corning</td>
			<td>36216004</td>
			<td>Dilute 1:100 in DPBS</td>
		</tr>
		<tr>
			<td>Cell Recovery Solution</td>
			<td>Corning</td>
			<td>354253</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (DPBS)</td>
			<td>HyClone</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Thermo Fisher Scientific</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (100X)</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin (50mg/mL stock)</td>
			<td>Life Technologies</td>
			<td>15750060</td>
			<td></td>
		</tr>
		<tr>
			<td>GDNF (10 &#181;g stock)</td>
			<td>Sigma</td>
			<td>SRP3200</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM stock)</td>
			<td>Life Technologies</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Mesh Celll Strainer (100 &#181;m)</td>
			<td>Fisher Scientific</td>
			<td>22363549</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Mesh Celll Strainer (40 &#181;m)</td>
			<td>Fisher Scientific</td>
			<td>22363547</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Serologic Pipet 5 ml</td>
			<td>Fisher Scientific</td>
			<td>13-678-11D</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA (1X)</td>
			<td>Life Technologies</td>
			<td>25200-056</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating enteric glial cells from the submucosa and lamina propria of a mouse

Source: Wang, Z. et al., <a href="https://app.jove.com/v/57629">Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse.</a>&#160;J. Vis. Exp. (2018).
This video demonstrates a method for isolating enteric glial cells from the submucosa and lamina propria of the mouse intestine. It outlines the steps involved in extracting submucosa and lamina propria tissue, isolating the enteric glial cells from them, and culturing the cells on a coated well-plate to establish an enteric glial cell culture.

<img alt="Figure 1" src="/files/ftp_upload/22551/22551_Figure1.jpg" /> 
Figure 1: Set up and preparation of mouse intestines.&#160;(A) Picture of isolation set up on ice including extracted intestines. (B) Picture of 5 mL-syringe attached to a 20 G blunt end needle to flush out fecal contents. (C) Engineered end of a wooden cotton swab (~4 cm) soaking in the DPBS buffer.
<p class="jove_content" fo:keep-together.w...

Isolating Enteric Glial Cells From the Submucosa and Lamina Propria of a Mouse

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Sterile ...

Take a mouse small intestine segment previously stripped of the longitudinal muscle and myenteric plexus, layers containing enteric neurons, and glial cells.
Cut up the tissue and place it in a buffer containing EDTA. Incubate with rocking.
EDTA disrupts cell-cell junctions, detaching the epithelial mucosa from the underlying lamina propria and submucosa, connective tissue layers containing enteric glial cells.
Pipette repeatedly to dislodge the epithelial cells.
Filter through a cell strainer and discard the flow-through.
Transfer the tissue to a non-enzymatic dissociation buffer and incubate it with rocking. The dissociation buffer loosens the lamina propria and submucosal extracellular matrix, detaching the cells.
Pipette repeatedly to further dissociate the cells.
Filter through a cell strainer to collect the cells.
Centrifuge and resuspend the cells in a buffer.
Take adhesion protein-coated wells containing a glial growth medium and plate the cells.
The coating facilitates cell adhesion, while the growth factors selectively promote glial cell proliferation, establishing a lamina propria and submucosal glial cell culture.

isolating-enteric-glial-cells-from-submucosa-lamina-propria

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

55 Aufrufe. Quelle: Wang, Z. et al., Isolierung enterischer Gliazellen aus der Submukosa und Lamina propria der erwachsenen Maus.J. Vis. Exp. (2018). Dieses Video zeigt eine Methode zur Isolierung enterischer Gliazellen aus der Submukosa und der Lamina propria des Mausdarms. Es werden die Schritte beschrieben, die bei der Extraktion von Submukosa- und Lamina propria-Gewebe, der Isolierung der enterischen Gliazellen und der Kultivierung der Zellen auf einer beschichteten Well-Platte zur Etablierung einer en...

Serie: Isolierung enterischer Gliazellen aus der Submukosa und der Lamina propria einer Maus - Konzept

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

Isolierung von Lamina Propria Mononukleären Zellen aus murine Colon mit Kollagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Forschung

JoVE Journal

Immunologie und Infektion

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Isolierung und Kultur der primären Maus Keratinozyten aus neonatalen und erwachsenen Maus Haut

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Biologie

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Isolierung, Charakterisierung und Differenzierung von kardialen Stammzellen von Erwachsenen Maus Herzen

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolating Enteric Glial Cells From the Submucosa and Lamina Propria of a Mouse

Transkript

Isolating Enteric Glial Cells From the Submucosa and Lamina Propria of a Mouse