Name: isolating and immunostaining lymphocytes and dendritic cells from murine peyer's patches
Uploaded: 2013-03-17T14:00:00
Description: Watch this Scientific Journal Video about Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches at JoVE.com

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. Iso...

<ol>
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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. 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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'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.

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

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