Name: unraveling key players of humoral immunity: advanced and optimized lymphocyte isolation protocol from murine peyer's patches
Uploaded: 2018-11-21T14:00:00
Description: 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

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 ...

<ol>
	<li>van den Berg, T. K., van der Schoot, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+&#8216;self&#8217;+recognition:+a+role+for+CD47-SIRP&#945;+interactions+in+hematopoietic+stem+cell+transplantation.">Innate immune &#8216;self&#8217; recognition: a role for CD47-SIRP&#945; interactions in hematopoietic stem cell transplantation.</a> Trends in Immunology. 29 (5), 203-206 (2008).</li><li>Mowat, A. M., Agace, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+specialization+within+the+intestinal+immune+system.">Regional specialization within the intestinal immune system.</a> Nature Reviews Immunology. 14 (10), 667-685 (2014).</li><li>Reboldi, A., Cyster, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peyer&#8217;s+patches:+Organizing+B-cell+responses+at+the+intestinal+frontier.">Peyer&#8217;s patches: Organizing B-cell responses at the intestinal frontier.</a> Immunological Reviews. 271 (1), 230-245 (2016).</li><li>Heel, K. A., McCauley, R. D., Papadimitriou, J. M., Hall, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Peyer&#8217;s+patches.">Review: Peyer&#8217;s patches.</a> Journal of Gastroenterology and Hepatology. 12 (2), 122-136 (1997).</li><li>Fagarasan, S., Kinoshita, K., Muramatsu, M., Ikuta, K., Honjo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+class+switching+and+differentiation+to+IgA-producing+cells+in+the+gut+lamina+propria.">In situ class switching and differentiation to IgA-producing cells in the gut lamina propria.</a> Nature. 413 (6856), 639-643 (2001).</li><li>Hopkins, S. A., Niedergang, F., Corthesy-Theulaz, I. E., Kraehenbuhl, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+recombinant+Salmonella+typhimurium+vaccine+strain+is+taken+up+and+survives+within+murine+Peyer&#8217;s+patch+dendritic+cells.">A recombinant Salmonella typhimurium vaccine strain is taken up and survives within murine Peyer&#8217;s patch dendritic cells.</a> Cellular Microbiology. 2 (1), 59-68 (2000).</li><li>Shreedhar, V. K., Kelsall, B. L., Neutra, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholera+toxin+induces+migration+of+dendritic+cells+from+the+subepithelial+dome+region+to+T-+and+B-cell+areas+of+Peyer's+patches.">Cholera toxin induces migration of dendritic cells from the subepithelial dome region to T- and B-cell areas of Peyer's patches.</a> Infection and Immunity. 71 (1), 504-509 (2003).</li><li>Sato, A., Iwasaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peyer&#8217;s+patch+dendritic+cells+as+regulators+of+mucosal+adaptive+immunity.">Peyer&#8217;s patch dendritic cells as regulators of mucosal adaptive immunity.</a> Cellular and Molecular Life Sciences. 62 (12), 1333-1338 (2005).</li><li>Bemark, M., Boysen, P., 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=Induction+of+gut+IgA+production+through+T+cell-dependent+and+T+cell-independent+pathways.">Induction of gut IgA production through T cell-dependent and T cell-independent pathways.</a> Annals of the New York Academy of Sciences. 1247 (1), 97-116 (2012).</li><li>Fagarasan, S., Kawamoto, S., Kanagawa, O., Suzuki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+Immune+Regulation+in+the+Gut:+T+Cell-Dependent+and+T+Cell-Independent+IgA+Synthesis.">Adaptive Immune Regulation in the Gut: T Cell-Dependent and T Cell-Independent IgA Synthesis.</a> Annual Review of Immunology. 28, 243-273 (2010).</li><li>Hase, K., 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=Uptake+through+glycoprotein+2+of+FimH+++bacteria+by+M+cells+initiates+mucosal+immune+response.">Uptake through glycoprotein 2 of FimH + bacteria by M cells initiates mucosal immune response.</a> Nature. 462 (7270), 226-230 (2009).</li><li>Wu, H., 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+Inhibitory+Role+for+the+Transcription+Factor+Stat3+in+Controlling+IL-4+and+Bcl6+Expression+in+Follicular+Helper+T+Cells.">An Inhibitory Role for the Transcription Factor Stat3 in Controlling IL-4 and Bcl6 Expression in Follicular Helper T Cells.</a> Journal of Immunology. 195 (5), 2080-2089 (2015).</li><li>Vinuesa, C. G., Tangye, S. G., Moser, B., Mackay, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follicular+B+helper+T+cells+in+antibody+responses+and+autoimmunity.">Follicular B helper T cells in antibody responses and autoimmunity.</a> Nature Reviews Immunology. 5 (11), 853-865 (2005).</li><li>Victora, G. D., Nussenzweig, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germinal+Centers.">Germinal Centers.</a> Annual Review of Immunology. 30, 429-457 (2012).</li><li>Vaeth, 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=Store-Operated+Ca2+Entry+in+Follicular+T+Cells+Controls+Humoral+Immune+Responses+and+Autoimmunity.">Store-Operated Ca2+Entry in Follicular T Cells Controls Humoral Immune Responses and Autoimmunity.</a> Immunity. 44 (6), 1350-1364 (2016).</li><li>Meli, A. P., 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=The+Integrin+LFA-1+Controls+T+Follicular+Helper+Cell+Generation+and+Maintenance.">The Integrin LFA-1 Controls T Follicular Helper Cell Generation and Maintenance.</a> Immunity. 45 (4), 831-846 (2016).</li><li>Fu, W., 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=Deficiency+in+T+follicular+regulatory+cells+promotes+autoimmunity.">Deficiency in T follicular regulatory cells promotes autoimmunity.</a> Journal of Experimental Medicine. 215 (3), 815-825 (2018).</li><li>Esp&#233;li, M., Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> T follicular helper cells - Methods and Protocols. , (2015).</li><li>Couter, C. J., Surana, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Flow+Cytometric+Characterization+of+Murine+Small+Intestinal+Lymphocytes.">Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes.</a> Journal of Visual Experiments. (111), e54114 (2016).</li><li>De Jesus, M., Ahlawat, S., Mantis, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolating+And+Immunostaining+Lymphocytes+and+Dendritic+Cells+from+Murine+Peyer&#8217;s+Patches.">Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer&#8217;s Patches.</a> Journal of Visual Experiments. (73), e50167 (2013).</li><li>Pastori, C., Lopalco, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+in+vitro+Activation+of+Mouse+Peyer&#8217;s+Patch+Cells+from+Small+Intestine+Tissue.">Isolation and in vitro Activation of Mouse Peyer&#8217;s Patch Cells from Small Intestine Tissue.</a> Bio-protocol. 4 (21), e1282 (2014).</li><li>Fukuda, S., Hase, K., Ohno, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+Mouse+Ligated+Peyer&#8217;s+Patch+Intestinal+Loop+Assay+to+Evaluate+Bacterial+Uptake+by+M+cells.">Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells.</a> Journal of Visual Experiments. (58), 3225 (2011).</li><li>Naito, Y., 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=Germinal+Center+Marker+GL7+Probes+Activation-Dependent+Repression+of+N-Glycolylneuraminic+Acid,+a+Sialic+Acid+Species+Involved+in+the+Negative+Modulation+of+B-Cell+Activation.">Germinal Center Marker GL7 Probes Activation-Dependent Repression of N-Glycolylneuraminic Acid, a Sialic Acid Species Involved in the Negative Modulation of B-Cell Activation.</a> Molecular and Cellular Biology. 27 (8), 3008-3022 (2007).</li><li>Bollig, N., 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=Transcription+factor+{IRF4}+determines+germinal+center+formation+through+follicular+T-helper+cell+differentiation.">Transcription factor {IRF4} determines germinal center formation through follicular T-helper cell differentiation.</a> Proceedings of the National Academy of Science of U. S. A. 109 (22), 8664-8669 (2012).</li><li>P&#233;rez-Mazliah, D., 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=Follicular+Helper+T+Cells+are+Essential+for+the+Elimination+of+Plasmodium+Infection.">Follicular Helper T Cells are Essential for the Elimination of Plasmodium Infection.</a> EBioMedicine. 24, 216-230 (2017).</li><li>Sage, P. T., Sharpe, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+follicular+regulatory+cells+in+the+regulation+of+B+cell+responses.">T follicular regulatory cells in the regulation of B cell responses.</a> Trends Immunology. 36 (7), 410-418 (2015).</li><li>Van Damme, N., 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=Chemical+agents+and+enzymes+used+for+the+extraction+of+gut+lymphocytes+influence+flow+cytometric+detection+of+T+cell+surface+markers.">Chemical agents and enzymes used for the extraction of gut lymphocytes influence flow cytometric detection of T cell surface markers.</a> Journal of Immunological Methods. 236 (1-2), 27-35 (2000).</li><li>Meenan, 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=Altered+expression+of+alpha+4+beta+7,+a+gut+homing+integrin,+by+circulating+and+mucosal+T+cells+in+colonic+mucosal+inflammation.">Altered expression of alpha 4 beta 7, a gut homing integrin, by circulating and mucosal T cells in colonic mucosal inflammation.</a> Gut. 40 (2), 241-246 (1997).</li><li>Cao, A. 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>

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 ...

Research

JoVE Journal

Cancer Research

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 ...

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. ...

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. ...

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 ...

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

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 ...

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 ...

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

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 ...

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

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 ...

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 ...

Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor ...