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 &#38; 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 &#181;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 ...

unraveling-key-players-humoral-immunity-advanced-optimized-lymphocyte

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

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

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of acute leukemia. However, the nature of this disease has not been well documented in the literature. Here, we provide a technical protocol for characterizing C1498 cells in vitro and for determining the nature of the induced leukemia in vivo. The first part of this procedure is focused on determining the hematopoietic lineage and the stage of differentiation of cultured C1498 cells. To achieve this, multi-parametric flow cytometric staining is used to detect hematopoietic cell markers. Immunofluorescence microscopy, cytochemistry and a May-Gr&#252;nwald Giemsa staining are then performed to assess the expression of myeloperoxidase, the activity of esterases and cellular morphology, respectively. The second part of this protocol is dedicated to describing the leukemia disease that is induced in vivo. The latter can be achieved by determining the frequencies of leukemic and inherent cells in the blood, hematopoietic organs (e.g., bone marrow and spleen) and non-lymphoid tissues (e.g., the liver and lungs) using specific staining and flow cytometry analyses. The nature of the leukemia is then confirmed using May-Gr&#252;nwald Giemsa staining and staining for specific esterases in the bone marrow. Here, we present the results that were obtained using this protocol in age-matched C1498- and PBS-injected mice.

This manuscript provides a technical procedure that can be used to characterize C1498 cell cultures in vitro and the acute leukemia induced in mice after their injection. Phenotypic and functional analyses are performed using flow cytometry, immunofluorescence microscopy, cytochemistry and May-Gr&#252;nwald Giemsa staining.

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

One-step Protocol for Evaluation of the Mode of Radiation-induced Clonogenic Cell Death by Fluorescence Microscopy

Research on ionizing radiation (IR)-induced clonogenic cell death is important for understanding the effect of IR on malignant tumors and normal tissues. Here, we describe a quick and cost-effective one-step assay for simultaneously assessing the major modes of clonogenic cell death induced by IR, i.e., apoptosis, mitotic catastrophe, and cellular senescence. In this method, cells grown on a cover slip are irradiated with X-rays and stained with 4&#39;,6-diamidino-2-phenylindole dihydrochloride (DAPI). Using fluorescence microscopy, apoptosis, mitotic catastrophe, and cellular senescence are identified based on the characteristic morphologies of the DAPI-stained nuclei. Apoptosis is determined by the presence of apoptotic bodies (i.e., condensed and fragmented nuclei). Mitotic catastrophe is determined by the presence of nuclei that exhibit two or more distinct lobes and micronuclei. Cellular senescence is determined by the presence of senescence-associated heterochromatic foci (i.e., nuclear DNA containing 30-50 bright, dense foci). This approach allows the experimenter to easily screen for clonogenic cell death modes using various cell lines, treatment settings, and/or time points, with the goal of elucidating the mechanisms of cell death in the target cells and conditions of interest.

Research on ionizing radiation (IR)-induced clonogenic cell death is important for understanding the effects of IR on malignant tumors and normal tissues. Here, we describe a one-step assay for assessing the major modes of IR-induced clonogenic cell death based on morphology of 4&#39;,6-diamidino-2-phenylindole dihydrochloride (DAPI)-stained nuclei visualized by fluorescence microscopy.

Research on ionizing radiation (IR)-induced clonogenic cell death is important for understanding the effect of IR on malignant tumors and normal tissues. ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising approach to cancer therapy. The glycosaminoglycan placental chondroitin sulfate A (plCSA) is expressed on a wide range of cancer cells and placental trophoblasts, and malarial protein VAR2CSA can specifically bind to plCSA. A reported placental chondroitin sulfate A binding peptide (plCSA-BP), derived from malarial protein VAR2CSA, can also specifically bind to plCSA on cancer cells and placental trophoblasts. Hence, plCSA-BP-conjugated nanoparticles could be used as a tool for targeted drug delivery to human cancers and placental trophoblasts. In this protocol, we describe a method to synthesize plCSA-BP-conjugated lipid-polymer nanoparticles loaded with doxorubicin (plCSA-DNPs); the method consists of a single sonication step and bioconjugate techniques. In addition, several methods for characterizing plCSA-DNPs, including determining their physicochemical properties and cellular uptake by placental choriocarcinoma (JEG3) cells, are described.

Synthesis and Characterization of Placental Chondroitin Sulfate A plCSA-Targeting Lipid-Polymer Nanoparticles

Here, we present a protocol for the synthesis of placental chondroitin sulfate A binding peptide (plCSA-BP)-conjugated lipid-polymer nanoparticles via single-step sonication and bioconjugate techniques. These particles constitute a novel tool for the targeted delivery of therapeutics to most human tumors and placental trophoblasts to treat cancers and placental disorders.

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising ...

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented with increasing depth, the incidence rate of cutaneous melanoma has shown a rapid and continuous increase during recent decades. In order to find effective preventative methods, it is important to understand the early steps of melanoma initiation in the skin. Previous data has demonstrated that follicular melanocyte stem cells (MCSCs) in the adult skin tissues can act as melanoma cells of origin when expressing oncogenic mutations and genetic alterations. Tumorigenesis arising from melanoma-prone MCSCs can be induced when MCSCs transition from a quiescent to active state. This transition in melanoma-prone MCSCs can be promoted by the modulation of either hair follicle stem cells&#39; activity state or through extrinsic environmental factors such as ultraviolet-B (UV-B). These factors can be artificially manipulated in the laboratory by chemical depilation, which causes transition of hair follicle stem cells and MCSCs from a quiescent to active state, and by UV-B exposure using a benchtop light. These methods provide successful spatial and temporal control of cutaneous melanoma initiation in the murine dorsal skin. Therefore, these in vivo model systems will be valuable to define the early steps of cutaneous melanoma initiation and could be used to test potential methods for tumor prevention.

The following procedure describes a method for spatial and temporal control of melanocytic tumor initiation in murine dorsal skin, using a genetically engineered mouse model. This protocol describes macroscopic as well as microscopic cutaneous melanoma initiation.

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

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

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Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples