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Immunology and Infection

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

Published: March 17th, 2013

DOI:

10.3791/50167

1Division of Infectious Diseases, 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.

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 μm 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 >2.5 x 106 cells with >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 μm) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

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), whic....

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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

  1. (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 μl per mouse.

2. Iso.......

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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.......

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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.......

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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 f....

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

Table 1. Reagents and equipment used in this study.

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