Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches

This method can answer key questions about the phenotype of mouse Peyer's Patch isolated T and B cell subsets. In particular, follicular T helper and germinal center B cell populations. The main advantage of this technique is that it dispenses with several laborous preparation steps such as collagenase based digestion that compromise the quality and identity of the isolated lymphocytes.

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 peritineal 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.

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 6-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, mesenteric side down, on a paper towel. Moisten the entire intestinal segment with RPMI 10%FBS to avoid tissue dehydration and stickiness and locate the Peyer'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's patches, use curved surgical scissors with the curve facing up to gently excise each patch from it's distal and proximal borders, taking care to exclude the surrounding intestinal tissue, and place the Peyer's patches into individual wells of a 12-well plate containing ice-cold RPMI 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 millimeter micro pipette tip so that the diameter is large enough to aspirate individual Peyer's patches and transfer the Peyer's patches into 150 millimeter conical tubes per mouse containing 25 milliliters of 37 degrees Celsius RPMI 10%FBS. Then place the tube in an orbital shaker at 37 degrees Celsius with continuous agitation at 125-150 RPM for 10 minutes. At the end of the agitation, transfer the Peyer's patches onto 140 micrometer cell strainer per mouse, and use the rubber end of one 10 milliliter syringe plunger per animal to gently disrupt the Peyer's patches through the mesh into individual 50 milliliter conical tubes.

Rinse the strainers with 15-20 milliliters of cold RPMI 10%FBS and collect the cells by centrifugation. After carefully discarding the supernatant, resuspend the cells at an approximately 1 times 10 to the 7th cells per milliliter of RPMI 10%FBS concentration for counting. Then transfer 2-2.5 times 10 to the 6th cells per 200 microliters of medium to each well of a 96-well round bottom plate and pellet the cells by centrifugation.

After discarding the supernatant with gentle flicking, centrifuge wash the cells in 200 microliters of standing buffer. Label the cells in 100 microliters of fixable viability dye, diluted in protein-free buffer for 30 minutes at 4 degrees Celsius, protected from light, followed by 2 washes and fresh staining buffer. After discarding the supernatant, resuspend the pellets in 20 microliters of FC block for 15 minute incubation at 4 degrees Celsius, followed by the addition of 80 microliters of primary surface antibody cocktail of interest, for 30 minutes on ice, protected from light.

At the end of the surface antibody incubation, centrifuge wash the cells two times in an excess volume of staining buffer, and resuspend the pellets in 100 microliters of secondary surface staining solution, supplemented with streptavidin. After 15 minutes on ice protected from light, centrifuge wash the cells 2 times in fresh staining buffer and resuspend the pellets in 200 microliters of fixation, permeablization working solution for a no more than 20 minute incubation on ice, protected from light. At the end of the incubation, centrifuge the plate immediately, followed by a centrifuge was in 200 microliters of permeablization buffer per well.

Next, resuspend the cells in 20 microliters of FC block, diluted in permeablization buffer as demonstrated, followed by the addition of 80 microliters of the intracellular antibody cocktail of interest, for 30 minutes at room temperature, protected from light. Wash the cells with 100 microliters of permeablization buffer, followed by a second wash in 200 microliters of permeablization buffer. Then, resuspend the pellets in 200 microliters of staining buffer, and transfer the cells to the appropriate corresponding 5 milliliter flow cytometric analysis tubes, in a final volume of 400 microliters of staining buffer per tube.

Peyer's patches are not evenly distributed throughout the small intestine, but are localized more densely towards the distal and proximal ends of the tissue. This protocol facilitates the isolation of a Peyer's patch lymphocyte population that demonstrates a forward-side scattered distribution, similar to that observed for splenocytes, with a greater than 95%cell viability. CD4 CD19 Double Positive cells constitute about 1%of the total Peyer's patch lymphocyte population.

These express high levels of CXCR5 and GL7, which, when not excluded, can lead to false positive results in the gating of T-helper follicular and germinal center B cell, respectively. Compared to other secondary lymphoid organs, Peyer's patches exhibit a significantly higher ratio of germinal center B cells, at a range of 2-10%of the total B cell population under steady state conditions. Like germinal center B cells, the follicular T-helper cell fraction also varies in individual unimmunized mice, with a range of 10-20%of total, the CD4 Positive Peyer's patch T-lymphocyte population.

Collagenase based enzymatic digestion leads to massive reduction of CXCR5 T-helper follicular cell expression, while leaving the expression of other surface molecules unaffected. After this development, this technique paved the way for the identification of key cellular factors within the Peyer's patches that are involved in mucosal and humeral immunity.