We would like to thank Laura Strauss and Peter Sage for helpful discussions and support with flow cytometry analyses.

All studies and experiments described in this protocol were conducted under guidelines according to Institutional Animal Care and Use Committee (IACUC) of Beth Israel Deaconess Medical Center.
1. Designing Experimental Set-up and Mouse Groups
<ol>
	<li>(Optional) Co-house the experimental mice to facilitate horizontal transmission of gut microbiota between experimental mice and to reduce non-specific variability within PP lymphocytes. Additionally, use littermate controls of the same gender to minimize variability.</li>
</ol>
2. 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 backside down and sanitize the abdomen with 70% ethanol. Perform a laparotomy by cutting the abdominal skin and peritoneum along the midline from 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 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 abdomen.</li>
		<li>Pinpoint 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 pylorus and duodenum, and snip the duodenum at this level, which will lead to complete detachment of 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 place 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 site of SI will stick to the paper towel keeping the anti-mesenteric site facing up.</li>
		<li>Identify the PPs, which appear as white multi-lobulated aggregates in a &#8220;cauliflower-like&#8221; 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 visual identification of PPs.</li>
		<li>After identifying PPs on the anti-mesenteric side, place the surgical curved-end scissor on PPs (curve should face up) restraining the PP from its distal and proximal border. 
		Optional:&#160;Push the PP gently toward the blades of scissor 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 C57BL/6 mouse, 5-10 PPs (average size, multi-lobulated) can be collected. By aiming 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 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 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 1 mL pipette. Aspirate the PPs with 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 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. 
		NOTE: (i) Prior to cell counting, the total cell number for each mouse PP group can be approximately estimated using the following formula: &#8220;0.5-1 x 106 cells x (number of PPs) = total cell count&#8221;. Further dilutions with trypan blue for cell counting might be necessary. (ii) As an alternative to manual counting, automated cell counters can be used.</li>
		<li>Transfer 2-2.5 x 106 cells in appropriate volume (e.g., 200 &#181;L) into a 96-well round-bottom plate. 
		NOTE: For single color and negative control samples, 0.5-1 x 106 cells might be sufficient.</li>
		<li>Centrifuge the plate at 350 x g for 5 min at 4 &#176;C. Flick the plate.</li>
		<li>Wash the cells in 200 &#181;L of Staining Buffer.</li>
	</ol>
	</li>
</ol>
3. Surface Antibody Staining
<ol>
	<li>Viability Staining:
	<ol>
		<li>After the final wash, resuspend the cells in 100 &#956;L of fixable viability dye diluted in PBS (1:1,000). Incubate for 30 min on ice or at 4 &#176;C in the dark. 
		NOTE: (i) Intracellular staining for the detection of key transcription factors such as Foxp3 or Bcl-6 requires fixation of the cells. In that case, non-fixable viability dyes (e.g., 7-AAD, DAPI) cannot be used. (ii) To dilute fixable viability dye, do not use any staining buffer that contains protein. The media used in this step must be protein-free. (iii) Exclusion of dead cells by using viability staining is crucial because dead cells can cause serious technical difficulties in flow cytometry analysis by emitting autofluorescence and by binding surface antibodies nonspecifically, which might lead to false positive results.</li>
		<li>Wash the cells twice with staining buffer. Centrifuge at 350 x g for 5 min at 4 &#176;C. Flick the plate.</li>
	</ol>
	</li>
	<li>Fc Block and Surface -Layer I:
	<ol>
		<li>Prepare the Fc-block solution by diluting anti-CD16/32 antibody (1:200) in staining&#160;buffer.</li>
		<li>Resuspend the cells in 20 &#956;L of prepared Fc-block solution. Incubate for 15 min on ice.</li>
		<li>Without washing, add 80 &#956;L of surface antibody cocktail (see Table 1 for the antibody summary) prepared at appropriate dilutions. Incubate on ice for at least 30 min.</li>
		<li>Wash twice by adding excessive staining&#160;buffer. Centrifuge at 350 x g for 5 min at 4 &#176;C. Flick the plate.</li>
	</ol>
	</li>
	<li>Surface -Layer II:
	<ol>
		<li>Prepare Streptavidin staining solution by diluting fluorochrome-conjugated Streptavidin in staining buffer.</li>
		<li>After the final wash, resuspend the cells with 100 &#956;L of pre-diluted Streptavidin (1:100) staining solution. Incubate for at least 15 min on ice in the dark.</li>
		<li>Wash twice with staining buffer. Centrifuge at 350 x g for 5 min at 4&#176;C. Flick the plate. 
		Optional: If intracellular staining for follicular&#160;regulatory T cell detection is not desired, after the last wash, resuspend the cells in 200 &#956;L of staining buffer and transfer into appropriate tubes to acquire data in flow cytometer. When the samples are not fixed, acquisition of data by flow cytometry should be performed within 3&#8211;4 h to obtain accurate results.</li>
	</ol>
	</li>
</ol>
4. Cell Fixation
<ol>
	<li>Prepare fixation/permeabilization (Fix/Perm) working solution using the reagents from Foxp3/Transcription Factor Staining Buffer Set. Mix one-part of fixation/permeabilization concentrate with three parts of fixation/permeabilization diluent to the desired final volume.</li>
	<li>After the final wash, resuspend the cells in 200 &#956;L of Fix/Perm working solution.</li>
	<li>Incubate the plate on ice or at 4 &#176;C for 20 min. Do not exceed 20 min for this step. Longer incubation time might severely decrease the cell recovery.</li>
	<li>Centrifuge at 350 x g for 5 min at 4 &#176;C. Flick the plate. (Optional) After this step, fixed cells can be stored for several days at 4 &#176;C in staining buffer containing bovine serum albumin (BSA) or FBS until subsequent intracellular staining or flow cytometry acquisition.</li>
	<li>Resuspend the cells in 200 &#956;L of permeabilization Buffer (x1) freshly pre-diluted in purified deionized water.</li>
	<li>Centrifuge at 350 x g for 5 min at room temperature&#160;(RT).</li>
	<li>Wash once with 200 &#956;L of permeabilization buffer and centrifuge at 350 x g for 5 min at RT.</li>
</ol>
5. Intracellular Staining
<ol>
	<li>Prepare the Fc-block solution by diluting anti-CD16/32 antibody (1:200) in permeabilization buffer. 
	NOTE: After the fixation step, the cells must be maintained in permeabilization buffer until the end of the intracellular staining process.</li>
	<li>After the final wash, resuspend the cells in 20 &#956;L of Fc-block solution. Incubate for 10&#8211;15 min at RT in the dark.</li>
	<li>Without washing, add 80 &#956;L of intracellular antibody cocktail (100 &#956;L final volume) pre-diluted in permeabilization buffer. Incubate for 30 min at RT.</li>
	<li>Add 100 &#956;L of Perm Buffer, and centrifuge at 350 x g for 5 min at RT.</li>
	<li>Wash once with 200 &#956;L of permeabilization buffer, centrifuge at 350 x g for 5 min at RT.</li>
	<li>After the final wash, resuspend the cells in 200 &#956;L of staining buffer and transfer the cells into appropriate tubes (final volume of 400 &#956;L in staining buffer) and acquire data by flow cytometry. Samples stained in 96-well plate may also be&#160;run without transferring into tubes if a flow cytometer with plate reader option is available. This step minimizes cell loss during cell transferring.</li>
	<li>Acquire a minimum of 5 x 105 total cells on the flow cytometer to be able to perform reproducible analysis of TFH, TFR as well as GC B cells.</li>
</ol>

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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin+(IL)+-21+promotes+intestinal+IgA+response+to+microbiota.">Interleukin (IL) -21 promotes intestinal IgA response to microbiota.</a> Mucosal Immunology. 8 (5), 1072-1082 (2015).</li><li>Wei, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+enforces+functional+integrity+of+regulatory+T+cells+by+coupling+environmental+cues+and+metabolic+homeostasis.">Autophagy enforces functional integrity of regulatory T cells by coupling environmental cues and metabolic homeostasis.</a> Nature Immunology. 17 (3), 277-285 (2016).</li><li>Autengruber, A., Gereke, M., Hansen, G., Hennig, C., Bruder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+enzymatic+tissue+disintegration+on+the+level+of+surface+molecule+expression+and+immune+cell+function.">Impact of enzymatic tissue disintegration on the level of surface molecule expression and immune cell function.</a> European Journal of Microbiology &amp; Immunology. 2 (2), 112-120 (2012).</li><li>Trapecar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Optimized+and+Validated+Method+for+Isolation+and+Characterization+of+Lymphocytes+from+HIV++Human+Gut+Biopsies.">An Optimized and Validated Method for Isolation and Characterization of Lymphocytes from HIV+ Human Gut Biopsies.</a> AIDS Research and Human Retroviruses. 33 (S1), (2017).</li><li>Bergqvist, P., Gardby, E., Stensson, A., Bemark, M., Lycke, N. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+IgA+Class+Switch+Recombination+in+the+Absence+of+CD40+Does+Not+Occur+in+the+Lamina+Propria+and+Is+Independent+of+Germinal+Centers.">Gut IgA Class Switch Recombination in the Absence of CD40 Does Not Occur in the Lamina Propria and Is Independent of Germinal Centers.</a> Journal of Immunology. 177 (11), 7772-7783 (2006).</li><li>Keil, B., Gilles, A. M., Lecroisey, A., Hurion, N., Tong, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+of+collagenase+from+Achromobacter+iophagus.">Specificity of collagenase from Achromobacter iophagus.</a> FEBS Letters. 56 (2), 292-296 (1975).</li><li>Mora, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+imprinting+of+gut-homing+T+cells+by+Peyer&#8217;s+patch+dendritic+cells.">Selective imprinting of gut-homing T cells by Peyer&#8217;s patch dendritic cells.</a> Nature. 424 (6944), 88-93 (2003).</li><li>Reboldi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+immunology:+IgA+production+requires+B+cell+interaction+with+subepithelial+dendritic+cells+in+Peyer&#8217;s+patches.">Mucosal immunology: IgA production requires B cell interaction with subepithelial dendritic cells in Peyer&#8217;s patches.</a> Science. 352 (6287), (2016).</li></ol>

No conflicts of interest declared.

Here, we describe a protocol optimized for flow cytometric characterization of TFH and GC B cells. One of the major advantages of our protocol is that it enables the isolation of up to 107 (average 4&#8211;5 x 106 cells) total PP cells from a single mouse (C57BL/6 strain) without any digestive process. We observed that the total cell yield was positively correlated with the number of PPs and could be estimated from the following simple equation which is helpful for experimental planning: &#34;total ...

The entire gastrointestinal tract from the beginning to the end is decked with an extensive lymphoid network that contains immune cells more than any other organ in human and mouse1. Peyer&#39;s patches (PPs) constitute a major component of the intestinal branch of this cellular immune organization, so-called gut-associated lymphoid tissue (GALT)2,3. Within PPs, thousands of millions of antigens derived from dietary materials, commensal microbiota and pathogens are being sampled continuously, and when necessary appropriate immune responses toward them are mounted thus maintaining intestinal immune homeostasis. In that sense, PPs could be named as &#34;tonsils of the small intestine&#34;. PPs consist of major sub-compartments: subepithelial dome (SED), large B-cell follicle zones; the overlying follicle-associated epithelium (FAE) and interfollicular region (IFR) where T cells are located4. This unique compartmentalization of PPs enables different effector cell subsets to cooperate, thus, confers immunocompetence in the gut.
PPs lack afferent lymphatics, and due to this reason, antigens transported to PPs from small intestine are not being conducted through lymphatic vessels in contrast to most of the other lymphoid organs. Instead, specialized epithelial cells located in FAE, so-called M cells, are responsible for the transfer of luminal antigens into the PPs5. Subsequently, the transported antigens are picked up by the dendritic cells (DCs) and phagocytes that are located in the subepithelial dome (SED) region beneath the FAE6,7. This antigen sorting process by DCs in the PP is crucial to initiate adaptive immune response8 and subsequent generation of IgA secreting cells9.
Due to the heavy antigenic burden from commensal flora and dietary material, PPs host endogenously activated effector T and B cell subsets in great abundances such as TFH and IgA+ GC B cells10, suggesting that PPs represent a site of active immune response11. Detection of up to 20&#8211;25% TFH cells within total CD4+ T cell compartment and up to 10&#8211;15% GC B cells within total B cells is possible in PPs collected from unimmunized young C57BL/6 mice12. In contrast to other T helper cell types (i.e., Th1, Th2, Th17 cells), TFH cells show unique tropism into B cell follicles primarily owing to CXCR5 expression, which promotes TFH cell homing along CXCL13 gradient13. In the B cell follicle zones of PPs, TFH cells induce IgA class switch recombination and somatic hypermutation in activated B cells from which high-affinity IgA producing cells differentiate14. Subsequently, these antibody-secreting plasma cells migrate to the lamina propria (LP) and regulate immune homeostasis in the gut10.
Identification and characterization of TFH and GC B cell populations within PPs might enable researchers to investigate the dynamics of humoral immune responses under steady-state conditions without the need of time-consuming immunization models traditionally used in TFH-GC B cell studies15,16,17,18. Analyzing TFH cells within PPs is not as straightforward as other cell subsets. Technical challenges include identifying ideal tissue preparation conditions, surface antibody-marker combination, as well as selecting appropriate positive and negative controls. Both TFH and PP research fields exhibit great variability in terms of experimental procedures and are far from conferring a consensus to establish standardized protocols due to several reasons. First, each cell subset within PPs tends to be affected differentially by tissue preparation conditions requiring further modifications in a cell subset-specific manner. Second, there is a significant discrepancy among the reported methods regarding the details of cell preparation from PPs. Third, the number of protocol-based comparative studies investigating ideal tissue preparation techniques and experimental conditions for PP and TFH research is rather limited.
Current protocol-based studies suggested for PP cell preparation19,20,21,22 were not TFH- or GC B cell-oriented. Moreover, some tissue preparation conditions recommended for PPs19,20 such as collagenase-based digestion were found to affect the outcome of TFH identification by flow cytometry negatively18. On this basis, we reasoned that an optimized, standardized and reproducible protocol that can be used to study TFH and GC B cell dynamics within PPs would be valuable to investigators working on this topic. This need gave us the impetus to generate an improved and up-to-date protocol for the isolation and characterization of PP lymphocytes that is finely optimized for cellular recovery, viability, and efficiency for flow cytometric characterization of several T and B cell subsets. We also aimed to exclude several laborious preparation steps suggested in previous protocols, thereby, reducing the required manipulations and time for tissue and cell preparation from PPs.

<table><tbody><tr><td>anti-mouse CD4 antibody</td><td>eBioscience, Biolegend*</td><td>17-0041-81 ,10054*</td><td>For detailed information see Table 1</td></tr><tr><td>anti-mouse CD19 antibody</td><td>eBioscience</td><td>MA5-16536</td><td>For detailed information see Table 1</td></tr><tr><td>anti-mouse PD-1 antibody</td><td>eBioscience</td><td>61-9985-82</td><td>For detailed information see Table 1</td></tr><tr><td>anti-mouse ICOS antibody</td><td>eBioscience</td><td>12-9942-82</td><td>For detailed information see Table 1</td></tr><tr><td>anti-mouse GL7 antibody</td><td>Biolegend</td><td>144610</td><td>For detailed information see Table 1</td></tr><tr><td>anti-mouse CXCR5 antibody</td><td>Biolegend*, BD Bioscience</td><td>145512*, 551960</td><td>For detailed information see Table 1</td></tr><tr><td>anti-mouse BCL-6 antibody</td><td>Biolegend</td><td>358512</td><td>For detailed information see Table 1</td></tr><tr><td>anti-mouse Foxp3 antibody</td><td>eBioscience</td><td>17-5773-82</td><td>For detailed information see Table 1</td></tr><tr><td>Streptavidin-BV421</td><td>BD Bioscience</td><td>563259</td><td>For detailed information see Table 1</td></tr><tr><td>FixableViability Dye</td><td>eBioscience</td><td>L34957</td><td>For detailed information see Table 1</td></tr><tr><td>7AAD</td><td>Biolegend</td><td>420404</td><td>For detailed information see Table 1</td></tr><tr><td>FcBlock (CD16/32)</td><td>BD Bioscience</td><td>553141</td><td>For detailed information see Table 1</td></tr><tr><td>Collagenase II</td><td>Worthington</td><td>LS004176</td><td></td></tr><tr><td>Collagenase IV</td><td>Worthington</td><td>LS004188</td><td></td></tr><tr><td>Foxp3/Transcription Factor Staining Buffer Set</td><td>eBioscience</td><td>00-5523-00</td><td></td></tr><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>

unraveling key players of humoral immunity: advanced and optimized lymphocyte isolation protocol from murine peyer's patches

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense against pathogens. It is well established that Peyer&#39;s patches (PPs) have an essential role in the mucosal immune network by hosting several effector T and B cell subsets. A certain fraction of these effector cells, follicular T helper (TFH) and germinal center (GC) B cells are professionalized in the regulation of humoral immunity. Hence, the characterization of these cell subsets within PPs in terms of their differentiation program and functional properties can provide important information about mucosal immunity. To this end, an easily applicable, efficient and reproducible method of lymphocyte isolation from PPs would be valuable to researchers. In this study, we aimed to generate an effective method to isolate lymphocytes from mouse PPs with high cell yield. Our approach revealed that initial tissue processing such as the use of digestive reagents and tissue agitation, as well as cell staining conditions and selection of antibody panels, have great influence on the quality and identity of the isolated lymphocytes and on experimental outcomes.
Here, we describe a protocol enabling researchers to efficiently isolate lymphocyte populations from PPs allowing reproducible flow cytometry-based assessment of T and B cell subsets primarily focusing on&#160;TFH and GC B cell subsets.

In contrast to a previous protocol20, we have observed that PPs are not evenly distributed throughout the SI but are localized more densely towards the distal and proximal ends of the SI as shown in Figure 1A. Flow cytometric analysis showed that, if followed correctly, our protocol gives a PP lymphocyte population that demonstrates forward-side scatter distribution similar to splenocytes (<strong clas...

Watch this Scientific Journal Video about Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches at JoVE.com

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

In this study, we present a novel and effective protocol for the isolation of lymphocytes from Peyer&#39;s Patches (PPs), which can be subsequently used for in vivo and in vitro functional assays as well as flow cytometric studies of follicular T helper and germinal center B cells.

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense ...

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13.5K Views.  Harvard Medical School. In this study, we present a novel and effective protocol for the isolation of lymphocytes from Peyer's Patches (PPs), which can be subsequently used for in vivo and in vitro functional assays as well as flow cytometric studies of follicular T helper and germinal center B cells.

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

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Immunology and Infection

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Biology

Analysis of Somatic Hypermutation in the JH4 intron of Germinal Center B cells from Mouse Peyer's Patches

Within the germinal centers of lymphoid organs, mature B cells alter their expressed immunoglobulin (Ig) by introducing untemplated mutations into the variable coding exons of the Ig heavy and light chain gene loci. This process of somatic hypermutation (SHM) requires the enzyme activation-induced cytidine deaminase (AID), which converts deoxycytidines (C), into deoxyuridines (U). Processing the AID-generated U:G mismatches into mutations by the base excision and mismatch repair pathways introduces new Ig coding sequences that may produce a higher affinity Ig. Mutations in AID or DNA repair genes can block or significantly alter the types of mutations observed in the Ig loci. We describe a protocol to quantify JH4 intron mutations that uses fluorescence activated cell sorting (FACS), PCR, and Sanger sequencing. Although this assay does not directly measure Ig affinity maturation, it is indicative of mutations in Ig variable coding sequences. Additionally, these methods utilize common molecular biology techniques which&#160;analyze&#160;mutations in Ig sequences of multiple B cell clones. Thus, this assay is an invaluable tool in the study of SHM and Ig diversification.

Presented here is an assay to quantify somatic hypermutation within the immunoglobulin heavy chain gene locus using germinal center B cells from mouse Peyer&#8217;s patches.

Within the germinal centers of lymphoid organs, mature B cells alter their expressed immunoglobulin (Ig) by introducing untemplated mutations into the ...

Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important phenomenon in the gut immunity. The stroma of secondary lymphoid organs, such as Peyer&#8217;s patches (PPs) or mesenteric lymph nodes, are where na&#239;ve lymphocytes sense antigens. Na&#239;ve lymphocytes circulate through the bloodstream to reach high endothelial venules, the portal of entry into PPs. Some immunomodulators are estimated to influence lymphocyte migration, but the precise evaluation of microcirculation dynamics is very difficult, and establishing a method to observe lymphocyte migration in vivo can contribute to the clarification of the precise mechanisms. We refined the method of collecting lymphocytes from the lymph duct and observing the detailed dynamics of gut-tropic lymphocytes in rat PPs. We chose confocal laser scanning microscopy to observe rat PPs in vivo and recorded it using time-lapse photography. We can now obtain clear images that can contribute to the analysis of lymphocyte dynamics.

Here, we describe a precise method for collecting thoracic duct lymphocytes and observing the migration of gut-tropic lymphocytes in rat Peyer&#8217;s patches for 3 hours using time-lapse photography. This technique can clarify how the dynamics of lymphocytes are affected under inflammatory conditions.

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important ...