Fluorescence Microscopy and Flow Cytometric Analysis of Peyer's Patches and Intestinal Immune Cells

María C. López1

1 Wadsworth Center, Albany, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 18.13
DOI:  10.1002/0471140856.tx1813s33
Online Posting Date:  August, 2007
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Abstract

In recent years researchers have become more aware of the importance of the gut‐associated immune system since it is in direct interaction with the entry site for virus, bacteria, and all type of food contaminants, including numerous toxins that can alter mucosal immunity. Peyer's patches (PP) are considered the inductive site for protein antigen presentation in the gut as well as the starting point for IgA B‐cell differentiation. The IgA found in feces comes from IgA secreted by IgA lamina propria lymphocytes (LPL), and its presence is a sign of normal physiology, in that IgA plays a role in absorption and immune defense against gut‐associated pathogens. Methods presented in this unit are intended to analyze PP and intestinal intraepithelial and LPL to determine whether the complexity of the mucosal‐associated lymphoid tissue and its components have been altered by any form of external damage. The protocols explain how to isolate and culture isolated cells; how to stain and analyze; and also how to cryopreserve the gut. Curr. Protoc. Toxicol. 33:18.13.1‐18.13.20. © 2007 by John Wiley & Sons, Inc.

Keywords: Peyer's patches; intestinal intraepithelial lymphocytes; intestinal lamina propria lymphocytes; mucosal immunology; IgA; T lymphocytes; cytokines; flow cytometry; fluorescence microscopy

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Mononuclear Cell Suspensions from Peyer's Patches
  • Support Protocol 1: Preparation of Cytospins of PP Cells for Immunomicroscopy
  • Support Protocol 2: Staining of PP Cells on Cytospins for Immunomicroscopy
  • Support Protocol 3: Lymphocyte Culture for Intracellular Cytokine and Surface Marker Simultaneous Determination
  • Support Protocol 4: Staining for Surface Markers and Intracellular Cytokines
  • Basic Protocol 2: Isolation of Intestinal Intraepithelial and Lamina Propria Lymphocytes
  • Alternate Protocol 1: Isolation of Intestinal LPL
  • Support Protocol 5: Preparation of Mesenteric Lymphocyte Controls for Flow Cytometric Analysis of Intestinal Lymphoid Cells
  • Support Protocol 6: Preparing Tissues for Freezing, Cutting in a Cryostat, and Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Mononuclear Cell Suspensions from Peyer's Patches

  Materials
  • Mice, 4‐ to 20‐weeks‐old
  • Anesthetic of choice (pentobarbital, ketamine/xylazine, or carbon dioxide)
  • 70% ethanol
  • Hanks' balanced salt solution (Sigma)
  • PBS/2% (v/v) FBS or RPMI/2% (v/v) FBS, optional
  • Complete medium (see recipe)
  • Dissecting instruments: scissors, forceps
  • 35‐mm tissue culture dishes (small)
  • 15‐ml conical tubes (Falcon)
  • 40‐µm cell strainers (Falcon)
  • Small petri dish
  • 3‐ to 5‐ml syringe plunger
  • Refrigerated centrifuge
  • Additional reagents and equipment for counting cells ( appendix 3B)

Support Protocol 1: Preparation of Cytospins of PP Cells for Immunomicroscopy

  Materials
  • Peyer's patch cell suspension ( protocol 1)
  • Complete medium (see )
  • 90% methanol
  • Cytospin cytocentrifuge and applicator (Shandon)
  • SuperFrost glass slides
  • Additional reagents and equipment for preparing a Peyer's patch cell suspension ( protocol 1)

Support Protocol 2: Staining of PP Cells on Cytospins for Immunomicroscopy

  Materials
  • Cytospins ( protocol 2)
  • Phosphate buffered saline (PBS), pH 7.4 (Invitrogen)
  • PBS/2% (w/v) bovine serum albumin (BSA)
  • Primary antibody (see Table 18.13.1)
  • PBS/0.01% (v/v) Tween
  • Fluorochrome‐conjugated secondary antibody or biotinylated antibody (see Table 18.13.1)
  • PBS/glycerol 10:90
  • ImmEdge hydrophobic barrier pen (Vector)
  • Coplin staining jars
  • Humidified chamber
  • Coverslip
  • Transparent nail polish
  • Additional reagents and equipment for obtaining cytospins ( protocol 2)
    Table 8.3.1   Materials   Reagents Used to Analyze Peyer's Patches Cytospins and Small Intestine Frozen Sections a   Reagents Used to Analyze Peyer's Patches Cytospins and Small Intestine Frozen Sections

    Primary reagents
    Antigen Antibody Supplier
    Primary antibodies
    IgM (µ chain) Biotinylated rat anti–mouse Serotec; http://www.serotec.com/index.php
    IgA (α chain) FITC‐conjugated Southern Biotechnology; http://www.southernbiotech.com/
    DAPI Molecular Probes, Invitrogen; http://probes.invitrogen.com/
    Cytokeratin 8/18 Biotinylated mouse anti–human (cross reacts with mouse) Novocastra, distributed by Visionbiosystems; http://www.vision‐bio.com/
    CD3 Biotinylated hamster anti–mouse Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/
    CD4 Purified or biotinylated rat anti–mouse Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/ Caltag/Invitrogen; http://www.caltag.com/home.asp
    CD8α Purified or biotinylated rat anti–mouse Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/ Caltag/Invitrogen; http://www.caltag.com/home.asp
    CD8β Purified or biotinylated rat anti–mouse Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/ Caltag/Invitrogen; http://www.caltag.com/home.asp
    αβ‐TCR Purified or biotinylated hamster anti–mouse Caltag/Invitrogen; http://www.caltag.com/home.asp
    γδ‐TCR Purified or biotinylated hamster anti–mouse Caltag/Invitrogen; http://www.caltag.com/home.asp
    Secondary reagents
    Texas Red conjugated donkey anti–rat Jackson ImmunoResearch; http://www.jacksonimmuno.com/
    FITC conjugated goat anti‐hamster Jackson ImmunoResearch; http://www.jacksonimmuno.com/
    FITC conjugated streptavidin Jackson ImmunoResearch; http://www.jacksonimmuno.com/
    Texas Red conjugated streptavidin Jackson ImmunoResearch; http://www.jacksonimmuno.com/
    AMCA conjugated streptavidin Jackson ImmunoResearch; http://www.jacksonimmuno.com/
    Streptavidin Vector; http://www.vectorlabs.com/
    Biotinylated goat anti‐streptavidin Vector; http://www.vectorlabs.com/

     aAntibodies listed in Table 18.13.1 are used to analyze cells on tissue sections under a fluorescence microscope by the naked eye. This analysis requires the use of a secondary antibody to enhance the signal. The researcher should establish the appropriate dilution for each antibody.

Support Protocol 3: Lymphocyte Culture for Intracellular Cytokine and Surface Marker Simultaneous Determination

  Materials
  • Lymphocytes ( protocol 1 or protocol 62)
  • Complete medium (see recipe) with 50 µM 2‐mercaptoethanol (2‐ME)
  • Phorbol 12‐myristate 13‐acetate (PMA)
  • Ionomycin
  • Brefeldin A (Epicentre Technology)
  • Tissue culture
  • 24‐well plates
  • 37°C incubator

Support Protocol 4: Staining for Surface Markers and Intracellular Cytokines

  Materials
  • Incubated PP cells ( protocol 4)
  • PBS/2% (w/v) BSA/0.1% (w/v) sodium azide (see recipe)
  • Brefeldin A
  • 4% (w/v) paraformaldehyde, pH 7.4 (see recipe)
  • PBS/2% (w/v) BSA/0.5% (w/v) saponin/0.1% (w/v) sodium azide (see recipe)
  • Fluorochrome‐conjugated antibodies that recognize mouse cytokines (Table 18.13.2)
  • Antibodies recognizing specific surface markers
  • 2% (w/v) paraformaldehyde, pH 7.4 (see recipe)
  • 96‐well V‐bottom plate
  • Vortex
  • Disposable 1.2‐ml microtiter tubes in racks resembling 96‐well plates (Life Science)
  • Falcon 12 × 75–mm “flow” tubes
  • Flow cytometer
  • Additional reagents and solutions for incubated PP cells ( protocol 4)
    Table 8.3.2   Materials   Reagents Used to Analyze Peyer's Patches and Small Intestine Isolated Cells b   Reagents Used to Analyze Peyer's Patches and Small Intestine Isolated Cells

    Antigen Antibody Supplier
    Surface membrane c
    αβ‐TCR Fluorochrome conjugated hamster anti–mouse Caltag/Invitrogen; http://www.caltag.com/home.asp Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/
    γδ‐TCR Fluorochrome conjugated hamster anti–mouse Caltag/Invitrogen; http://www.caltag.com/home.asp Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/
    CD2 Fluorochrome conjugated rat anti–mouse Serotec; http://www.serotec.com/index.php
    CD3 Fluorochrome conjugated hamster anti–mouse Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/
    CD4 Fluorochrome conjugated rat anti–mouse Caltag/Invitrogen; http://www.caltag.com/home.asp Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/
    CD5 Fluorochrome conjugated rat anti–mouse Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/
    CD8α Fluorochrome conjugated rat anti–mouse Caltag/Invitrogen; http://www.caltag.com/home.asp Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/
    CD8β Fluorochrome conjugated rat anti–mouse Caltag/Invitrogen; http://www.caltag.com/home.asp Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/
    CD19 Fluorochrome conjugated rat anti–mouse Caltag/Invitrogen; http://www.caltag.com/home.asp Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/
    CD23/IgE Fc receptor Fluorochrome conjugated rat anti–mouse Southern Biotechnology; http://www.southernbiotech.com/
    CD25 Fluorochrome conjugated rat anti–mouse Caltag, Invitrogen; http://www.caltag.com/home.asp Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/
    CD44 Fluorochrome conjugated rat anti–mouse Caltag/Invitrogen; http://www.caltag.com/home.asp Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/
    CD45 Fluorochrome conjugated rat anti–mouse Caltag/Invitrogen; http://www.caltag.com/home.asp Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/
    CD45Rb Fluorochrome conjugated rat anti–mouse Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/
    CD62L Fluorochrome conjugated rat anti–mouse Caltag/Invitrogen; http://www.caltag.com/home.asp Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/
    CD90 (Thy 1) Fluorochrome conjugated rat anti–mouse Caltag/Invitrogen; http://www.caltag.com/home.asp Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/
    CD117 (c‐kit) Fluorochrome conjugated rat anti–mouse Caltag/Invitrogen; http://www.caltag.com/home.asp Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/
    CD49b (DX5) Fluorochrome conjugated rat anti–mouse Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/
    NK1.1 Fluorochrome conjugated mouse anti–mouse Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/
    Cytoplasmic markers
    IFN‐γ Fluorochrome conjugated rat anti–mouse Caltag, Invitrogen; http://www.caltag.com/home.asp Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/
    IL‐2 Fluorochrome conjugated rat anti–mouse Caltag/Invitrogen; http://www.caltag.com/home.asp Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/
    IL‐4 Fluorochrome conjugated rat anti–mouse Caltag/Invitrogen; http://www.caltag.com/home.asp Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/
    IL‐5 Fluorochrome conjugated rat anti–mouse Caltag/Invitrogen; http://www.caltag.com/home.asp Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/
    IL‐7 Carrier free goat anti–mouse c R&D Systems; http://www.rndsystems.com/
    IL‐10 Fluorochrome conjugated rat anti–mouse Caltag, Invitrogen; http://www.caltag.com/home.asp Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/
    TNF‐α Fluorochrome conjugated rat anti–mouse Caltag/Invitrogen; http://www.caltag.com/home.asp Pharmingen, BD Biosciences; http://www.bdbiosciences.com/home/

     bAntibodies listed in Table 18.13.2 are used to study isolated cells that after staining will be analyzed in a flow cytometer. The researcher should establish the appropriate dilution for each antibody.
     cCan be conjugated with FITC (Sigma) or Alexa Fluor dyes (Molecular Probes, Invitrogen).

Basic Protocol 2: Isolation of Intestinal Intraepithelial and Lamina Propria Lymphocytes

  Materials
  • Percoll (Amersham)
  • 10× Phosphate‐buffered saline (PBS; Invitrogen)
  • 1× PBS (Invitrogen)
  • Dissected small intestines
  • Hanks' balanced salt solution (calcium‐ and magnesium‐free: modified HBSS; Sigma), ice cold
  • 1 mM DTT
  • Complete medium (see recipe)
  • PBS/0.5% (v/v) heat‐inactivated fetal bovine serum (HI FBS)
  • 100‐mm petri dish
  • 50‐ml conical tubes (Falcon)
  • Bacterial shaker
  • 50‐ml syringe
  • Sterile cotton/nylon wool columns for filtration or 70‐µm pore cell strainers (Falcon 2350)

Alternate Protocol 1: Isolation of Intestinal LPL

  Materials
  • Intestinal pieces ( protocol 6)
  • Hanks' balanced salt solution (HBSS; Sigma)
  • Collagenase D (final concentration 1 mg/ml, stock 100 mg/ml, in water; Roche)
  • DNase I (final concentration 0.1 mg/ml, stock 10 mg/ml in water; Roche)
  • Complete medium (see recipe)
  • PBS/0.5% (v/v) heat‐inactivated fetal bovine serum (HI FBS)
  • 50‐ml conical tubes (Falcon)
  • Bacterial shaker
  • Sterile cotton/nylon wool columns for filtration
  • Additional reagents and equipment for obtaining intestinal pieces ( protocol 6), culture of lymphocytes ( protocol 4), and staining for markers ( protocol 5)

Support Protocol 5: Preparation of Mesenteric Lymphocyte Controls for Flow Cytometric Analysis of Intestinal Lymphoid Cells

  Materials
  • Dry ice
  • Acetone
  • Isopentane
  • Intestinal pieces ( protocol 6)
  • Tissue Tek OCT compound
  • Styrofoam box
  • Stainless‐steel bowl
  • 150‐ml stainless steel beaker
  • Disposable base molds, Fisher Tissue Path
  • Long forceps
  • Aluminum foil
  • −80°C freezer
  • Cryostat
  • SuperFrost plus glass slides
  • Coplin jar
  • Additional reagents and equipment for isolation of intestinal sections ( protocol 6)
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Figures

Videos

Literature Cited

Literature Cited
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