We thank Renjie Song (Wadsworth Center Flow Cytometry Core) for assistance in cell analysis and Helen Johnson (Wadsworth Center Animal Histopathology Core) for preparation of paraffin sections. We thank Dr. Richard A. Cole (Wadsworth Center Light Microscopy Core) for assistance with confocal microscopy and image collection. We would like to acknowledge Andy Bentley (Wadsworth Center Photo and Illustration) for assistance with animations.
	MDJ is supported by the Life Sciences Research Foundation, Howard Hughes Medical Institute (HHMI) Fellowship. SA is supported by a Wadsworth Center-Health Research Inc. intramural postdoctoral fellowship. This work was supported in part by NIH grants HD061916 and GM082978.

Animals were housed under conventional, specific pathogen-free conditions and were treated in full compliance with the Wadsworth Center's Institutional Animal Care and Use Committee (IACUC) guidelines.
 1. Oral Gavage
 <ol>
 <li>(Optional) Gavage mouse strain of choice with antigen or microbes of interest using a 22 G x1.5-in. blunt-end feeding needle (Popper Scientific, New Hyde Park, NY). Delivery volumes should not exceed 400 &mu;l per mouse.</li>
 </ol>
 2. Isolation of PP Cells for Flow Cytometry
 <ol>
 <li>Euthanize mice by CO2 asphyxiation, according to institutional animal care and use committee (IACUC) guidelines.</li>
 <li>Cleanse the abdomen with 70% ethanol or betadine prior to surgery. Perform a standard laparotomy that involves a single (1 cm) incision using surgical grade scissors along the midline beginning about 1.5 cm from the base of the rib cage. Expose the peritoneal cavity and identify the cecum. Snip the terminal small intestine at the ileal-cecal junction and gently remove the intestine in its entirety. Take care not to hyperextend the intestinal tissue as it is being removed from the peritoneal cavity.</li>
 <li>Lay the small intestine on a bed of moist paper towels or Kimwipes. Wet the small intestine gently with saline to prevent tissue dehydration. Visually identify individual PPs located on the anti-mesenteric side of the intestine (Figure 1). Typically, a single mouse has 5 to 10 visible PPs that are evenly distributed from the duodenum (proximal) to ileum (distal). On average, four mice will yield 23 PPs.</li>
 <li>Using curved surgical scissors, gently excise individual PPs and place them in cold Hank's Balanced Salt Solution (HBSS). Note, the scissors should be placed curve-side up just above the PP and then gently applied to the tissue. Excise only the PP (not surrounding tissue) and transfer to cold HBSS. 
 Note: All incubations and centrifugation steps from this point forward in Section 2 should be done at 4 &deg;C to preserve cell viability.</li>
 <li>Transfer PPs into 5 ml Spleen Dissociation Medium and incubate for 15-20 min at 37 &deg;C while vigorously shaking at 250 rpm. Longer incubation time will negatively affect cell viability.</li>
 <li>To generate a single cell suspension, place PPs on a sterile (autoclaved) 70 &mu;m nylon mesh cell strainer and forcibly grind the tissue into the mesh using the base of a plunger from a 1 cc syringe. Alternatively, sandwich PPs between two frosted sterile glass microscope slides (autoclaved in envelopes) and gently grind tissues with a back and forth motion. Use a sterile transfer pipette to transfer the cell suspension into a 5 ml Falcon tube.</li>
 <li>Add EDTA to a final concentration of 1 mM to the cell suspension. Incubate on a rocker for 5 min at room temperature.</li>
 <li>Decant cell suspension through a second 70 &mu;m cell strainer to remove any remaining cellular aggregates or tissue debris. Collect cells in a 15 or 50 ml conical tube.</li>
 <li>Subject cells to gentle centrifugation (5 min at 500 x g). Decant supernatant and resuspend cells in flow buffer, which is phosphate buffered saline (PBS) containing 1% fetal calf serum (FCS).</li>
 <li>To determine cell number and viability, dilute cells 1:10 into trypan blue and visually inspect cells using a light microscope and a hemocytometer. Alternatively, a Countess cell counter (Invitrogen) or similar instrument can be used to automatically enumerate cell numbers and viability. 
 Starting with 20-25 PPs, this protocol should yield &gt;2.5 x 106 total cells. This equates to ~ 0.8-1.2 x 106 cells per mouse. </li>
 </ol>
 Antibody Labeling of Cells for Flow Cytometry
 <ol start="11">
 <li>Dispense cells (105 per well) into wells of a 96-well round bottom plate.</li>
 <li>Subject plate to gentle centrifugation (5 min at 1,000 x g), and decant supernatant by gently inverting the plate.</li>
 <li>Resuspend cells in Fc block buffer and incubate on ice for 15 min. While there are a number of commercially available Fc block buffers (e.g. rat anti-mouse CD16/CD32, BD Biosciences), we simply use spent medium from a rat B cell hybridoma (ATCC 2.4.G2) that secretes a monoclonal IgG1 against murine Fc&gamma; receptors for this step.</li>
 <li>Subject plate to gentle centrifugation (5 min at 1,000 x g) as above, and decant supernatant by gently inverting the plate.</li>
 <li>Add fluorophore-conjugated antibodies directly to cell suspensions at desired dilution, and incubate on ice for 30 min with continuous rocking.</li>
 <li>Subject plate to gentle centrifugation (5 min at 1,000 x g), and decant supernatant by gently inverting the plate.</li>
 <li>Wash cells with 100 &mu;l of flow buffer.</li>
 <li>Centrifuge plate at 1,000 x g for 5 min, and decant supernatant by gently inverting the plate.</li>
 <li>Resuspend cells 400 &mu;l fixation buffer, which consists of 300 &mu;l of flow buffer and 100 &mu;l of 1% paraformaldehyde in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 4 mM MgCl2 at pH 6).</li>
 <li>Subject cells to flow cytometry using a FACSCalibur or equivalent. Analyze results using Cell Quest Pro software version 5.2.</li>
 </ol>
 3. Preparation of PP Cryosections
 <ol>
 <li>In advance of tissue collection, add Optimal Cutting Temperature (OCT) compound to 7x7x5 mm plastic base molds. Also prepare a pool of liquid nitrogen (&gt;750 ml) in a dewar or ice bucket.</li>
 <li>Euthanize mouse and perform a laparotamy, as described above. Remove intestine and place on wet paper towels.</li>
 <li>To isolate PPs for cyrosectioning, cut 0.5 cm transverse segments of small intestine containing a single PP and transfer tissue to a Petri dish containing PBS. Gently rinse the lumen of these segments with PBS to remove unwanted fecal debris.</li>
 <li>Place intestinal tissue segments vertically inside base molds containing OCT. Immerse the molds in liquid nitrogen and allow tissue to freeze completely (1-2 min). The color of the OCT will change from clear to white when the tissue is fully frozen. For a more gradual cooling (e.g. to reduce cracking of the OCT), immerse mold in a bath of isopentane cooled with liquid nitrogen vapors. Note: Wear appropriate safety goggles when working with liquid nitrogen. </li>
 <li>Remove frozen base molds from the liquid nitrogen using forceps and allow the mold to warm at room temperature just long enough (~10-15 sec) to allow the edges of the OCT block to soften. To then remove the frozen block of OCT from the mold, invert the mold and press gently on the back to eject the block onto a clean 4 x 4 cm piece of aluminum foil. Immediately wrap the tissue in the foil and place in a labeled specimen bag stored in liquid nitrogen or on dry ice. Alternatively, transfer tissue in a freezer (&lt;-20 &deg;C). Use the tissue within a week, otherwise the blocks will become brittle with age.</li>
</ol>
Cryosectioning
 <ol start="6"> 
 <li>Cryosection the tissue using a Leica CM3050S cryomicrotome or equivalent. The chamber temperature on the cryostat should be set to -21 &deg;C.</li>
 <li>Strive for sections that are 12-14 &mu;m in thickness.</li>
 <li>Collect sections onto Fisher SuperFrost Plus (or equivalent) glass microscope slides and inspect visually using a standard low power light microscope. If multiple sections are being collected on a slide, ensure that they are clustered together for downstream staining applications.</li>
 <li>Store the slides at room temperature in a slide box and, ideally, use them within a few days.</li>
</ol>
Antibody Labeling of Cryosections and Confocal Microscopy Analysis
<ol start="10">
 <li>Immerse slides in acetone (or methanol) in tissue staining jar or racks for 2 min. Prolonged incubation may cause tissues to detach from the slide.</li>
 <li>Transfer slides to new staining jar and wash 3 times with PBS-T (1X PBS with 0.05% Tween-20) for 3 min each.</li>
 <li>Encircle the sections with an ImmEdge hydrophobic pen. This will ensure that reagents and antibodies will stay confined to that area during incubations.</li>
 <li>Incubate slides in block buffer (2% goat serum in PBS) for 30 min at 37 &deg;C in a humidified chamber, protected from light. </li>
 <li>Incubate slides with Fc block buffer (see above) for 10 min at 37 &deg;C. </li>
 <li>Dip slides in PBS-T and dry area around sections using Kimwipes or other absorbent tissue. Take care not to touch the actual tissue sections.</li>
 <li>Overlay tissue sections with primary antibody solution and incubate for 30 min at 37 &deg;C in a humidified chamber. Primary antibody is typically diluted in block buffer to a final concentration of 20 &mu;g/ml. The use of directly-labeled primary antibodies is desirable, because as many as three antibodies can be combined directly at this step. Directly-labeled antibodies also eliminate the need for additional antibody incubation steps. </li>
 <li>Wash slides 3 times (5 min each) by immersion in PBS.</li>
 <li>If necessary, incubate slides with relevant secondary antibodies for 30 min at 37 &deg;C in a moisture chamber.</li>
 <li>Wash slides 3 times (5 min each) by immersion in PBS.</li>
 <li>Incubate slides for 4 min in 1% paraformaldehyde in PHEM buffer, as described above.</li>
 <li>Rinse slides with PBS for 1 min.</li>
 <li>Dry area around sections using Kimwipes or other absorbent tissue. Take care not to touch the actual tissue sections. Using a pipette or dropper, gently place a drop of Prolong Gold mounting medium directly onto the section. Apply a glass coverslip onto the mounting medium taking care to avoid bubbles. Use blotting paper to absorb any excess mounting medium.</li>
 <li>Seal coverslips to microscope slides with commercial nail polish and allow to air dry. </li>
 <li>View slides with a Leica TCS SP5 or equivalent confocal microscope. </li>
 </ol>

<ol>
	<li>Mantis, N. J., Rol, N., Corthesy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretory+IgA's+complex+roles+in+immunity+and+mucosal+homeostasis+in+the+gut.">Secretory IgA's complex roles in immunity and mucosal homeostasis in the gut.</a> Mucosal. Immunol. 4, 603-611 (2011).</li><li>Neutra, M., Mantis, N., 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=Collaboration+of+epithelial+cells+with+organized+mucosal+lymphoid+tissue.">Collaboration of epithelial cells with organized mucosal lymphoid tissue.</a> Nature Immunology. 2, 1004-1009 (2001).</li><li>Rescigno, M., Sabatino, A. D. i. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+in+intestinal+homeostasis+and+disease.">Dendritic cells in intestinal homeostasis and disease.</a> J. Clin. Invest. 119, 2441-2450 (2009).</li><li>Suzuki, K., Kawamoto, S., Maruya, M., Fagarasan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GALT:+organization+and+dynamics+leading+to+IgA+synthesis.">GALT: organization and dynamics leading to IgA synthesis.</a> Adv. Immunol. 107, 153-185 (2010).</li><li>Iwasaki, A., Kelsall, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+distinct+Peyer's+patch+dendritic+cell+subsets+and+their+recruitment+by+chemokines+macrophage+inflammatory+protein+(MIP)-3alpha,+MIP-3beta,+and+secondary+lymphoid+organ+chemokine.">Localization of distinct Peyer's patch dendritic cell subsets and their recruitment by chemokines macrophage inflammatory protein (MIP)-3alpha, MIP-3beta, and secondary lymphoid organ chemokine.</a> Journal of Experimental Medicine. 191, 1381-1394 (2000).</li><li>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=Mucosal+dendritic+cells.">Mucosal dendritic cells.</a> Annu. Rev. Immunol. 25, 381-418 (2007).</li><li>Lelouard, H., Fallet, M., de Bovis, B., Meresse, S., Gorvel, 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=Peyer's+Patch+Dendritic+Cells+Sample+Antigens+by+Extending+Dendrites+Through+M+Cell-Specific+Transcellular+Pores.">Peyer's Patch Dendritic Cells Sample Antigens by Extending Dendrites Through M Cell-Specific Transcellular Pores.</a> Gastroenterology. 42, 592-601 (2011).</li><li>Lelouard, 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=Pathogenic+bacteria+and+dead+cells+are+internalized+by+a+unique+subset+of+Peyer's+patch+dendritic+cells+that+express+lysozyme.">Pathogenic bacteria and dead cells are internalized by a unique subset of Peyer's patch dendritic cells that express lysozyme.</a> Gastroenterology. 138, 173-184 (2010).</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> Infect. Immun. 71, 504-509 (2003).</li><li>Favre, L., Spertini, F., Corthesy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretory+IgA+possesses+intrinsic+modulatory+properties+stimulating+mucosal+and+systemic+immune+responses.">Secretory IgA possesses intrinsic modulatory properties stimulating mucosal and systemic immune responses.</a> J. Immunol. 175, 2793-2800 (2005).</li><li>Kelsall, B. L., Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+populations+of+dendritic+cells+are+present+in+the+subepithelial+dome+and+T+cell+regions+of+the+murine+Peyer's+patch.">Distinct populations of dendritic cells are present in the subepithelial dome and T cell regions of the murine Peyer's patch.</a> J. Exp. Med. 183, 237-247 (1996).</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's+Patch+Intestinal+Loop+Assay+to+Evaluate+Bacterial+Uptake+by+M+cells.">Application of a Mouse Ligated Peyer's Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells.</a> J. Vis. Exp. (58), e3225 (2011).</li><li>Geem, D., Medina-Contreras, O., Kim, W., Huang, C. S., Denning, T. 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No conflicts of interest declared.

In this article, we have provided parallel protocols for preparing PP single cell preparations for flow cytometric and functional analysis and cryosections for immunostaining. Both methods are highly reproducible and readily accessible, provided a flow cytometer and cryostat are available. For first time investigators it should be pointed out that when compared to the spleen, total cell yields from PPs are relatively meager. Nonetheless, the protocol we outline generally yields between 0.8-1.2 x 106 total PP c...

Peyer's patches (PPs) are macroscopic aggregates of organized lymphoid follicles present throughout the small intestine of humans and mice (Figure 1) and constitute the primary sites at which mucosal immune responses are initiated against dietary antigens, commensal bacteria, microbial pathogens, and oral vaccines 1-4. Unlike other peripheral lymphoid tissues such as the mesenteric lymph nodes, PPs lack afferent lymphatics. As such, adaptive immune responses in PPs are driven in response to antigens derived from the intestinal lumen. The sampling of luminal antigens is accomplished the by the follicle-associated epithelium (FAE), which consists of both enterocytes and antigen-sampling cells known as M cells. Beneath the FAE, in the sub-epithelial dome (SED) region, lies a network of dendritic cells (DCs) intermingled with macrophages, B cells, and CD4+ T cells 5-9. At the core of each PP lymphoid follicle are follicular dendritic cells (FDCs) and a B cell-rich central germinal center, flanked by T cell-rich interfollicular zones. Antigen sampling by PPs results in the development of IgA+ B cell plasmablasts and CD4+ effector and memory cells that seed the surrounding lamina propria and provide immunity to a wide range to mucosal invaders.
 Dissecting the complex immunologic events associated with antigen sampling, processing, and presentation in PPs is a daunting task, considering that PP cells constitute only a tiny fraction of the total lymphoid cells in intestinal mucosa. To aid in the in vitro characterization of cells in this environment, we provide a protocol for preparing total mouse PP cells for flow cytometric and functional analysis, as well as a protocol for preparing PP cryosections for immunofluorescence microscopy and immunohistology. Our protocol for the isolation, characterization, and immunostaining of mouse PP cells is not novel per se, as evidenced by the fact that there are numerous references dating back more than 25 years that cite these techniques 5,6,9-11. Rather, our protocol provides a streamlined (and visual) method for investigators collecting PPs for the first time. The techniques we describe are easily mastered and readily yield large numbers of cells with &gt;90% cell viability. The cryosectioning protocol yields highly reproducible serial sections ideally suited for immunofluorescence staining and confocal imaging. Furthermore, our protocol complements two other recent JoVE articles. The first, by Fukuda and colleagues, describes the use of ligated ileal loop assays to assess the uptake of pathogenic bacteria by PP M cells 12. The other, by Geem and colleagues, describes the isolation and characterization of DCs and macrophages from the mouse intestinal mucosa, but explicitly excludes PPs from their analysis 13.

<table border="1">
	 <tbody>
 <tr>
 <td>Item </td>
 <td>Company</td>
 <td>Cat. # </td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>OCT Compound</td>
 <td>Tissue-Tek</td>
 <td>4583</td>
 <td></td>
 </tr>
 <tr>
 <td>7x7x5 mm Base Molds</td>
 <td>Fisherbrand</td>
 <td>22-363-552</td>
 <td></td>
 </tr>
 <tr>
 <td>ImmEdge Pen</td>
 <td>Vector Labs</td>
 <td>H-4000</td>
 <td></td>
 </tr>
 <tr>
 <td>Superfrost Plus Slides</td>
 <td>Thermo Scientific</td>
 <td>4951</td>
 <td></td>
 </tr>
 <tr>
 <td>Edge Rite Blade</td>
 <td>Thermo Scientific</td>
 <td>4280L</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-Mouse CD11c-PE</td>
 <td>eBioscience</td>
 <td>17-0114-82</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-Mouse CD45R/B220-APC</td>
 <td>BD Pharmigen</td>
 <td>553092</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-Mouse CD3-FITC</td>
 <td>BD Pharmigen</td>
 <td>561798</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-Mouse CD4-PE</td>
 <td>BD Pharmigen</td>
 <td>553652</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti- Mouse CD8-PE</td>
 <td>BD Pharmigen</td>
 <td>553032</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-Mouse CD19-PercP</td>
 <td>BioLegend</td>
 <td>115531</td>
 <td></td>
 </tr>
 <tr>
 <td>Hank's balanced salt solution (HBSS) without phenol red </td>
 <td>Fisher Scientific</td>
 <td>14175-079</td>
 <td></td>
 </tr>
 <tr>
 <td>70 &mu;m cell strainer</td>
 <td>BD Falcon</td>
 <td>352350</td>
 <td></td>
 </tr>
 <tr>
 <td>Spleen Dissociation Medium</td>
 <td>Stem Cell Technologies</td>
 <td>7915</td>
 <td></td>
 </tr>
 <tr>
 <td>Goat serum </td>
 <td>Invitrogen</td>
 <td>16210-072</td>
 <td></td>
 </tr>
 <tr>
 <td>Fc Block</td>
 <td>ATCC 2.4.G2</td>
 <td>HB-197</td>
 <td>Supes obtained from cell line 2.4.G2</td>
 </tr>
 <tr>
 <td>Curved Scissor</td>
 <td>F.S.T</td>
 <td>14061-09</td>
 <td></td>
 </tr>
 <tr>
 <td>Cryostat </td>
 <td>Leica</td>
 <td>3050S</td>
 <td></td>
 </tr>
 <tr>
 <td>FACS Calibur</td>
 <td>BD </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Countess Cell Counter</td>
 <td>Invitrogen</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Hematoxylin</td>
 <td>Richard Allan</td>
 <td>7211</td>
 <td></td>
 </tr>
 <tr>
 <td>Eosin</td>
 <td>Richard Allan</td>
 <td>71304</td>
 <td></td>
 </tr>
 <tr>
 <td>Formalin</td>
 <td>Starplex Scientific</td>
 <td>3661</td>
 <td></td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>Table 1. Reagents and equipment used in this study.</td>
 </tr>
	 </tbody>
 </table>

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.

Flow cytometric analysis of monodisperse suspensions of total PP cells reveals a clear distinction between good and poor cell preparations. In good cell preparations with over 80% viability, the vast majority of cells demonstrate high forward scatter (FSC), an indicator of high cell volume, and low side scatter (SSC), an indicator of low cell granularity (Figure 2A). In this experiment, we also intentionally prepared a "poor cell preparation" by incubating PP cells during the isolation steps at ro...

Watch this Scientific Journal Video about Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches at JoVE.com

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer&#039;s Patches

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.

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 ...

isolating-immunostaining-lymphocytes-dendritic-cells-from-murine

Research

JoVE Journal

Immunology and Infection

32.4K Views.  New York State Department of Health. 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.

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

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

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 ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during inflammatory responses. Unfortunately, study of pDC function is hindered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation predominantly favor the production of cDCs over pDCs. Here we present a unique approach to efficiently generate pDCs from common lymphoid progenitors (CLPs) in vitro. Specifically, the protocol described details how to purify CLPs from bone marrow and generate pDCs by coculturing with &#947;-irradiated AC-6 feeder cells in the presence of Flt3 ligand. A unique characteristic of this culture system is that the CLPs migrate underneath the AC-6 cells and become cobblestone area-forming cells, a critical step for expanding pDCs. Morphologically distinct DCs, namely pDCs and cDCs, were generated after approximately 2 weeks with a composition of 70-90% pDCs under optimal conditions. Typically, the number of pDCs generated by this method is roughly 100-fold of the number of CLPs seeded. Therefore, this is a novel system with which to robustly generate the large numbers of pDCs required to facilitate further studies into the development and function of these cells.

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

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 ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

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 ...