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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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. Isolation of PP Cells for Flow Cytometry

1. Euthanize mice by CO₂ asphyxiation, according to institutional animal care and use ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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.