Abstract
The lumen of gastrointestinal tract is exposed to several potentially pathogenic microorganisms, thus it is extremely relevant to understand how immunosurveilance can be established. Peyer’s Patches (PPs) are oval or round lymphoid nodules that protrude from the outer wall of the ileum portion of small intestine. PPs contain a high percentage of B and T lymphocytes, macrophages and dendritic cells. Here we summarize a protocol for isolation and culture of mouse PP cells, which can be used to get a better insight into immunopathologies of microbes and to evaluate immune responses elicited by mucosal vaccines.
Keywords: Mucosae, Peyer's Patch, Intestine
Materials and Reagents
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Mouse (e.g. Balb/c) 8-20 weeks old
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Sterile PBS without Ca/Mg (Euroclone, catalog number: ECB4004L )
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Pen/Strep: 100x solution of penicillin/streptomycin/fungizone (Life Technologies, Gibco®, catalog number: 15240-062 )
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Gentamycin (50 mg/ml solution) (Life Technologies, Gibco®, catalog number: 15750-037 )
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RPMI 1640 (with glutamine) (Lonza, catalog number: BE12-702F )
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Fetal bovine serum (FBS) (heat inactivated) (Lonza, catalog number: DE14-801F )
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0.4% trypan blue (Sigma-Aldrich, catalog number: T8154 )
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Phorbol 12-Myristate 13-Acetate (PMA) (Sigma-Aldrich, catalog number: P8139 - follow the manufacturer's instructions to prepare stock solutions)
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Ionomycin (Sigma-Aldrich, catalog number: I9657 - follow the manufacturer's instructions to prepare stock solutions)
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Common disinfectant (i.e. 70-90% ethanol, tincture of iodine)
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PBS + 2% Pen/Strep (see Recipes)
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RPMI + 2% Pen/Strep (see Recipes)
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Complete medium (see Recipes)
Equipment
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Soft wood tablet and pins
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Aluminium foils (or another smooth, non-absorbent surface)
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Three sterile small thin surgical scissors
Note: In particular one for cutting the skin, the second one for cutting the muscular wall and the intestine, and the last one for removing PPs.
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Four sterile small thin surgical tweezers, two straight and two curved (straight ones for cutting the skin, the muscular wall and the intestine; curved ones to expel faeces and to remove PPs)
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70 µm cell strainer (BD Biosciences, Falcon®, catalog number: 352350 )
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2.5 ml syringes
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15 and 50 ml centrifuge tubes
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24-well cell culture plates
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Refrigerated centrifuge
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Sterile flow hood
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Thermostatic water bath
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Inverted microscope for cell cultures
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37 °C, 5% CO2 cell culture incubator
Procedure

Figure 1. Diagram for the isolation of PP cells from small intestine. Modified from Lefrancois and Lycke (2001).
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Removal of small intestine and excision of Peyer’s patches
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Euthanize mouse by CO2 asphyxiation, following guide lines approved by
your Institutional Animal Care and Use Committee. Proceed as soon as
possible to the aseptic intestine explantation and PP dissociation to
obtain maximum cell viability.
Note: All subsequent steps should be performed in a sterile flow hood.
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Gently lay down the mouse on its back, on a soft wood surface, stretch the limbs and fix the four paws with pins.
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Clean the abdomen with the disinfectant.
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A pair of scissors and a straight tweezers are used to perform a
midline incision and retract the skin, then open the muscular wall with
another cutting along median axis with a new pair of scissors and
tweezers.
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Cut small intestine approximately 0.5 cm below the
stomach, draw out of peritoneal cavity unfolding it. Cut intestine about
1 cm above the caecum, then remove fat, mesenteric lymph nodes and
adjacent tissues.
Note: You can cut the intestine with the same scissors and tweezers used for the muscular wall.
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Place the intestine on an aluminium foil and continuously moisten with cold PBS + 2% Pen/Strep to prevent drying.
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Expel faeces by placing a curved tweezers flat on the surface of the
intestine and press down along the entire length. Press gently to avoid
breaking the intestine.
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Remove PPs by tightening the patch with a
new curved tweezers and cutting it with scissors, avoiding as much as
possible the surrounding intestinal wall. The patches appear as small
protruding whitish/greyish nodules (diameter 1-2 mm), similar to the
lymph nodes, embedded in the outer intestinal wall, which appears to be
yellow-brown. In general, five to ten PPs can be obtained from a single
mouse, depending of age and strain.
Notes:
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The presence of an
excessive amount of surrounding epithelium reduces the yield of
recovered cells, as it could prevent a good mechanical dissociation.
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The removal of the surrounding tissue is crucial if the enzymatic
dissociation is carried out; in this last case the intraepithelial cells
could contaminate the resulting PP-single cells.
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Immediately after excision, save all PPs in 1 ml cold RPMI + 2% Pen/Strep.
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Isolation of PP cells by mechanical dissociation
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Transfer all PPs in a 70 µm cell strainer placed over a 50 ml tube.
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Dissociate PPs using the plunger of a 2.5 ml syringe and gently crush
the patches forcing through the filter. During dissociation wash the
filter with 5 ml of cold RPMI + 2% Pen/Strep (medium flow through the
filter contains the single cells of PPs).
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Repeat step 2 twice
more, until they are no longer visible pieces of tissue. At this point,
the tube contain PP-single cells flowed through the filter and suspended
in 15 ml of RPMI + 2% Pen/Strep.
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Centrifuge at 400 x g, 4 °C for
10 min. Discard the supernatant, resuspend the cells and add 10 ml of
cold RPMI + 2% Pen/Strep.
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Repeat step 4 two more times. After the last centrifugation resuspend the cells in 1-2 ml of cold RPMI + 2% Pen/Strep.
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Count viable cells using trypan blue dye exclusion.
Notes:
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The variability in the number of cells recovered from a single mouse is
very high, mainly due to the mouse (strain and age), the time elapsed
between the euthanization and dissociation of PP, and the accuracy in
removing every single PP.
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The mechanical dissociation allows to
isolate mainly non-adherent cells, such as B and T lymphocytes. Adherent
cells including monocytes, dendritic and non-immune epithelial cells
appear to be present in small quantities since they require an enzymatic
treatment to be isolated. The phenotype of recovered PP cells can be
checked by FACS analysis.
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If needed, PP cells can be further
purified by sorting with magnetic beads or FACS [see Guo et al. (2008)
and Thornton (2003) in the reference section].
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In vitro activation of PP cells
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Centrifuge an appropriate amount of cells at 400 x g, at 4 °C for 10 min.
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Resuspend the pellet in complete medium at a concentration of 2 x 106 cells/ml and culture the PP cells in 24-well plate, 1 ml/well, in
presence of 10 ng/ml PMA and 500 ng/ml Ionomycin for at least 24 h, in a
37 °C and 5% CO2 incubator.
Note: Other activation protocols,
specific for B or T lymphocytes, can be used [see Li et al. (2006) and
Muul et al. (2011) in the references section].
Optionals:
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To
eliminate adherent cells, PP cells can be cultured for 2-4 h in Complete
Medium (see recipes) with PMA and Ionomycin without antibiotics. The
presence of adherent cells (monocytes and/or dendritic cells) may
differentially modulate the activation of B and T lymphocytes with a
consequent alterations of the immune responses.
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To avoid as much
as possible a bacterial contamination, PP cells are washed and cultured
for 24 h as describe above, in Complete Medium + PMA/Ionomycin, with 100
μg/ml Gentamycin instead of Penicillin/Streptomycin. The use of a
different antibiotic ensures the elimination of all kinds of bacteria.
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After 24 h (or more) the supernatants of cultures can be collected,
centrifuged at 400 x g, at 4 °C for 10 min to eliminate the cells, and
used to test the presence of specific antibodies or cytokines. Activated
cells can be harvested, washed by centrifugation at 4 °C for 10 min and
used in flow cytometry assays and sorting.
Representative data
FACS analysis of in vitro activated PP cells.
PPs were obtained from 6 Balb/c mice as previously published (Pastori et al., 2014). In detail, two different immunization protocols were used: 3 mice received one intranasal (i. n.), while the other 3 mice received one intraperitoneal (i. p.) immunization. Aluminium hydroxide was used as adjuvant in i. p. immunization. For immunization Flock House Virus (FHV) was used in which an external loop of CCR5 (a seven transmembrane protein, belonging to chemokine receptor family and coreceptor for HIV-1) was introduced. The detailed immunization protocol has been described in Pastori et al. (2014).
After excision, PPs were mechanically dissociated and PP cells were in vitro activated with PMA and Ionomycin, as describe above. After 24 h cells were harvested and stained with antibodies against some markers of B cell activation.
As shown in Figure 2, CD138 and CD40/CD19 expression indicate a B cell activated phenotype for both protocols.

Figure 2. Expression of CD19, CD40, CD138 and co-expression of CD19/CD40 on PP cells after 24 h of PMA/Ionomycin activation. Percentage of positive cells refers to lymphocytes (gated on FSC vs SSC plot). Mean and standard deviation are shown.
Notes
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Images and more detailed information about mouse necropsy, intestine explantation and Peyer’ patches can be found in Scudamore (2013).
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The yield of cells isolated from PPs or intestinal lymph nodes is time sensitive. Proceed with the isolation as quickly as possible to obtain better cell viability.
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The mechanical dissociation of PPs gives a lower yield compared to the enzymatic one, but it does not alter the expression of surface antigens, thus, it is better in the case of subsequent FACS analysis or in vitro assays. The mechanical dissociation also leads to a relative loss of adherent accessory cells, such monocytes and/or dendritic cells, therefore, for the isolation of these cell types the enzymatic dissociation is preferable.
Recipes
Note: Prepare and keep all solutions in a sterile condition.
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PBS + 2% Pen/Strep
For 100 ml combine 98 ml of PBS and 2 ml of Pen/Strep
Refrigerate at 4 °C before use
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RPMI + 2% Pen/Strep
For 100 ml combine 98 ml of RPMI and 2 ml of Pen/Strep
Refrigerate at 4 °C before use
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Complete medium
For 100 ml combine 89 ml of RPMI (with Glutamine), 10 ml of heat inactivated FBS and 1 ml of Pen/Strep
Warm at 37 °C in a water bath before use
Acknowledgments
We thank Maria Rescigno for her help in mouse PPs isolation. This work was supported by Italian Ministry of Health, grant 40H15.
References
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Lefrancois, L. and Lycke, N. (2001). Isolation of mouse small intestinal intraepithelial lymphocytes, Peyer's patch, and lamina propria cells. Curr Protoc Immunol Chapter 3: Unit 3 19.
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Pastori, C., Diomede, L., Venuti, A., Fisher, G., Jarvik, J., Bomsel, M., Sanvito, F. and Lopalco, L. (2014). Induction of HIV-Blocking Anti-CCR5 IgA in Peyers's Patches without Histopathological Alterations. J Virol 88(7): 3623-3635.
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Pastori, C. and Lopalco, L. (2014). Isolation and
in vitro Activation of Mouse Peyer’s Patch Cells from Small Intestine Tissue.
Bio-protocol 4(21): e1282. DOI:
10.21769/BioProtoc.1282.