isolation of lymphocytes from a mouse peyer&#39;s patch

Isolation of Lymphocytes from a Mouse Peyer&#39;s Patch

Begin by placing a mouse in the supine position, and sterilizing the abdomen with 70% ethanol. Make a midline abdominal incision through the skin and peritoneum from the pubis to the rib cage to open the peritoneal cavity and locate the cecum and the ileocecal junction.
Make as distal as possible cut, at the junction to separate the small intestine from the cecum, and cut the mesentery with scissors to carefully remove the entire small intestine up to the pyloric sphincter.
Snip the junction between the pylorus and the duodenum to completely detach the small intestine from the abdominal cavity, and place the isolated small intestine into one well of a six-well plate containing cold RPMI medium, supplemented with 10% Fetal Bovine Serum or FBS on ice. Then, gently agitate the tissues manually until all of the segments are submerged in the cold medium.
When all of the intestines have been harvested, use forceps to grasp the mesenteric fat of one edge of the intestine and place the sample &#8212; mesenteric side down &#8212; on a paper towel. Moisten the entire intestinal segment with RPMI plus 10% FBS to avoid tissue dehydration and stickiness, and locate the Peyer&#39;s patches, which appear as white multilobulated aggregates with a cauliflower-like shape on the anti-mesenteric side of the intestinal wall.
To harvest the Peyer&#39;s patches, use curved surgical scissors with the curve facing up to gently excise each patch from its distal and proximal borders, taking care to exclude the surrounding intestinal tissue, and place the Peyer&#39;s patches into individual wells of a 12-well plate containing ice-cold RPMI plus 10% FBS, on ice, as they are collected.
When all of the patches have been acquired, use scissors to cut the tip of a 1-milliliter micropipette tip so the diameter is large enough to aspirate individual Peyer&#39;s patches, and transfer the Peyer&#39;s patches into 150-milliliter conical tube per mouse, containing 25-milliliters of 37 degrees Celsius RPMI plus 10% FBS.
Then, place the tubes in an orbital shaker at 37 degrees Celsius with continuous agitation at 125 to 150 RPM for 10 minutes. At the end of the agitation, transfer the Peyer&#39;s patches onto 140-micrometer cell strainer per mouse, and use the rubber end of 110-milliliter syringe plunger per animal to gently disrupt the Peyer&#39;s patches through the mesh into individual 50-milliliter conical tubes.
Rinse the strainers with 15 to 20 milliliters of cold RPMI plus 10% FBS, and collect the cells by centrifugation. After carefully discarding the supernatant, re-resuspend the cells at an approximately 1 x 107 cells per milliliter of RPMI plus 10% FBS concentration for counting. Then, transfer 2 to 2.5 x 106 cells per 200 micro-liters of medium, to each well of a 96-well round bottom plate, and pellet the cells by centrifugation.

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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. Surgical Excision and Tissue Preparation Steps:
<ol>
	<li>Surgical removal of the Small Intestine (SI)
	<ol>
		<li>Euthanize mice using CO2 asphyxiation or any equivalent method approved by the institutional animal ethics committee.</li>
		<li>Transfer the mouse to a dedicated area for surgical excision. Place the backside down and sanitize the abdomen with 70% ethanol. Perform a laparotomy by cutting the abdominal skin and peritoneum along the midline from the pubis to the rib cage thus opening the peritoneal cavity. 
		NOTE: Perform the first incision on a relatively small area of the skin to avoid penetrating the peritoneal cavity and damaging intestinal tissue. Continue the excision until the desired anatomical border.</li>
		<li>Identify the caecum, which is an ideal landmark for the detection of the terminal ileum, which constitutes the distal segment of the small intestine. 
		NOTE: Caecum is usually located on the lower left side of the mouse&#39;s abdomen.</li>
		<li>Pinpoint the ileocaecal junction and make a cut at this level as distal as possible to separate the small intestine from the caecum. Throughout the next steps, avoid excessive physical contact with the intestinal wall because fragile PPs collapse easily upon touch.</li>
		<li>Gently remove the entire small intestine until the pyloric sphincter by cutting the mesentery using scissors. Identify the junction between the pylorus and duodenum, and snip the duodenum at this level, which will lead to complete detachment of the small intestine from the abdominal cavity. 
		NOTE: (i) Avoid hyperextension as this might cause the rupture of the intestinal wall. (ii) If LP lymphocyte isolation is desired in addition to PP lymphocytes, complete removal of the mesenteric fat is necessary. However, for PP isolation only, remaining mesenteric fat could provide some benefits during the further isolation steps; therefore, it should be preserved.</li>
		<li>Place detached small intestines in a 6-well plate filled with cold RPMI + 10% fetal bovine serum (FBS) and gently agitate the tissues manually until all intestinal segments are submerged in the cold media. Maintain the tissues on ice throughout the next steps.</li>
		<li>After dissecting the desired number of mouse small intestines, proceed to PP excision from collected small intestines.</li>
	</ol>
	</li>
	<li>Surgical Excision of PPs and Preparation of Single Cell Suspension:
	<ol>
		<li>Gently transfer the small intestine on a paper towel by gripping the mesenteric fat using forceps and placing the mesenteric side facing the paper towel. Moisten the entire intestinal segment with cold RPMI + 10% FBS to avoid tissue dehydration and stickiness. 
		NOTE: Remaining mesenteric fat can be helpful at this stage because fat tissue segments on the mesenteric side of SI will stick to the paper towel keeping the anti-mesenteric side facing up.</li>
		<li>Identify the PPs, which appear as white multi-lobulated aggregates in a &#34;cauliflower-like&#34; shape on the anti-mesenteric side of the intestinal wall. 
		NOTE: Flushing out the luminal content is not recommended until all PPs are excised. Emptying the luminal content might cause the collapse of the PPs and will prevent the color contrast between PPs and the intestinal wall, which is very helpful for the visual identification of PPs.</li>
		<li>After identifying PPs on the antimesenteric side, place the surgical curved-end scissor on PPs (curve should face up) restraining the PP from its distal and proximal border. 
		Optional: Push the PP gently toward the blades of scissors using a fingertip. This maneuver will lead to the better exclusion of surrounding non-PP tissue. Excise the PPs gently, excluding the surrounding intestinal tissue. 
		NOTE: (i) This step is crucial to obtain maximal PP cell yield while minimizing the cell contamination from neighboring intestinal compartments such as LP and intestinal epithelium, which are also rich in T cells. (ii) From one SI excised from a C57BL/6 mouse, 5-10 PPs (average size, multi-lobulated) can be collected. By aiming for even smaller PPs (not multi-lobulated), collection of up to 12-13 PPs per mouse (C57BL/6) is possible using this protocol.</li>
		<li>Transfer the excised PPs to a 12-well tissue culture plate filled with ice-cold RPMI + 10% FBS and maintained on ice using forceps or curved surgical scissors. 
		NOTE: (i) Immediately after the excision of PPs, mucus, and intestinal content on the PP surface can be cleaned by rubbing the tissue gently on a paper towel. This step will help improve the viability of PP lymphocytes. (ii) Instead of transferring the PPs to a plate, transferring to separated tubes can also be considered depending on the total sample number.</li>
		<li>Optional: When PP excision and subsequent placement into the well plate are completed, the number and size of PPs collected from different experimental/mouse groups can be documented by taking a picture of the tissue culture plate containing PPs.</li>
		<li>Prepare a set of 50 mL conical tubes filled with 25 mL of RPMI + 10% FBS (pre-warmed at 37 &#176;C). Using a pair of scissors, cut the edge of a 1,000 &#181;L tip from the distance that will allow the aspiration of the PPs with a 1 mL pipette. Aspirate the PPs with a 1 mL pipette and transfer them from the 12-well tissue culture plate to the prepared 50 mL conical tubes. 
		NOTE: Use a new tip for each mouse sample to avoid cross-contamination among the samples.</li>
		<li>Secure the lid and place the tubes vertically in an orbital shaker at 37 &#176;C, with continuous agitation at 125&#8211;150 rpm for 10 min. Meanwhile, prepare a new set of 50 mL tubes and place a 40 &#181;m cell strainer on the top of each tube, through which single cell suspension will be prepared. 
		NOTE: (i) The agitation step will remove the remaining intestinal content, mucus, and cell debris, which decrease the cell viability and recovery of PP lymphocytes if not removed. (ii) Do not apply any kind of digestive enzymes on PP tissue because the digestion process causes a dramatic loss of CXCR5 expression from the cell surface.</li>
		<li>After the agitation, transfer the PPs to the 40 &#181;m cell strainer placed on the top of the newly prepared conical 50 mL tubes. Using the rounded side of a 10 mL syringe plunger, gently disrupt the PPs through the cell strainer to generate a single-cell suspension. Rinse the strainer with 15&#8211;20 mL of cold RPMI + 10 % FBS. 
		NOTE: (i) Before filtering, shake the tubes containing the PPs horizontally. This short shake will facilitate the transfer of PPs into cell strainers. (ii) Using a 70 &#181;m cell strainer for the isolation of non-lymphoid immune cells (e.g., monocytes, macrophages, DCs) is recommended.</li>
		<li>Centrifuge the single-cell suspensions at 350&#8211;400 x g for 10 min at 4 &#176;C.</li>
		<li>Carefully discard the supernatant and resuspend the cells at a concentration of 10 x 106 cells/mL. Count the cells using a hemocytometer.</li>
	</ol>
	</li>
</ol>

<table><tbody><tr><td>6-well, 12-well, &amp;amp; 96-well plates</td><td>Falcon/Corning</td><td>353046,353043/3596</td><td></td></tr><tr><td>50 ml conical tubes</td><td>Falcon</td><td>3520</td><td></td></tr><tr><td>40 &amp;micro;m cell strainer</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>10 ml syringe-plunger</td><td>Exel INT</td><td>26265</td><td></td></tr><tr><td>RPMI</td><td>Corning</td><td>15-040-CV</td><td></td></tr><tr><td>PBS</td><td>Corning</td><td>21-040-CM</td><td></td></tr><tr><td>FBS</td><td>Atlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td>Orbital shaker</td><td>VWR</td><td>Model 200</td><td></td></tr><tr><td>Curved-end scissor</td><td></td><td></td><td></td></tr><tr><td>Fine Serrated Forceps</td><td></td><td></td><td></td></tr><tr><td>Small curved scissor</td><td></td><td></td><td></td></tr></tbody></table>

Source: Yazicioglu, Y. F., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/58490">Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer&#39;s Patches.</a> J. Vis. Exp. (2018)
In this video, we demonstrate the isolation of lymphocytes from a harvested mouse Peyer&#39;s patch. The isolated lymphocytes can be used for further analysis.

Source: Yazicioglu, Y. F., et al. Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer&#39;s ...

Peyer&#39;s patches, PPs, aggregates of organized lymphoid tissues in the small intestinal wall, contain immune cells including B lymphocytes, T lymphocytes, dendritic cells, and macrophages.
To isolate lymphocytes from mouse PPs, begin with a euthanized mouse. Incise the abdominal wall, exposing the peritoneal cavity. Locate the cecum. Dissect the ileocecal junction to separate the cecum from the small intestine.
Excise the mesentery and incise the small intestine at the duodenum to remove the small intestine from the peritoneal cavity. Place the small intestine in a multi-well plate containing chilled medium and rinse to remove debris.
Position the small intestine with the mesenteric side facing down, and moisten with medium to maintain tissue hydration. Identify the PPs &#8212; white multi-lobulated aggregates on the anti-mesenteric side of the intestinal wall.
Carefully excise the PPs, excluding the surrounding intestinal tissue. Transfer to a tube containing medium. Incubate under constant agitation. This agitation step detaches the mucus, cell debris, and any fecal matter from the PPs, thus improving the recovery of PP lymphocytes.
Next, transfer the tissue into a cell strainer with an appropriate mesh size. Gently disrupt the PPs and release cells. Flush with medium through the strainer to obtain a single-cell lymphocyte suspension. Centrifuge the suspension at a low temperature to maintain the cell stability.
Resuspend the lymphocytes in medium, and use for further analysis.

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Isolation of Lymphocytes from a Mouse Peyer's Patch

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Isolation of Lymphocytes from a Mouse Peyer's Patch