Isolation of Lymphocytes from a Mouse Peyer's Patch

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:

1. Surgical removal of the Small Intestine (SI)
   1. Euthanize mice using CO₂ asphyxiation or any equivalent method approved by the institutional animal ethics committee.
   2. 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.
   3. 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's abdomen.
   4. 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.
   5. 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.
   6. 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.
   7. After dissecting the desired number of mouse small intestines, proceed to PP excision from collected small intestines.
2. Surgical Excision of PPs and Preparation of Single Cell Suspension:
   1. 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.
   2. Identify the PPs, which appear as white multi-lobulated aggregates in a "cauliflower-like" 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.
   3. 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.
   4. 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.
   5. 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.
   6. Prepare a set of 50 mL conical tubes filled with 25 mL of RPMI + 10% FBS (pre-warmed at 37 °C). Using a pair of scissors, cut the edge of a 1,000 µL 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.
   7. Secure the lid and place the tubes vertically in an orbital shaker at 37 °C, with continuous agitation at 125–150 rpm for 10 min. Meanwhile, prepare a new set of 50 mL tubes and place a 40 µm 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.
   8. After the agitation, transfer the PPs to the 40 µm 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 15–20 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 µm cell strainer for the isolation of non-lymphoid immune cells (e.g., monocytes, macrophages, DCs) is recommended.
   9. Centrifuge the single-cell suspensions at 350–400 x g for 10 min at 4 °C.
   10. Carefully discard the supernatant and resuspend the cells at a concentration of 10 x 10⁶ cells/mL. Count the cells using a hemocytometer.