Analysis and Purification of Mouse Intestinal Dendritic Cell and Macrophage Subsets by Flow Cytometry

Balázs Koscsó1, Milena Bogunovic1

1 Penn State University College of Medicine, Hershey, Pennsylvania
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.39
DOI:  10.1002/cpim.11
Online Posting Date:  August, 2016
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Abstract

The unit presents a method for analysis of intestinal dendritic cell (DC) and macrophage subsets by flow cytometry in the single cell suspension prepared from the mouse small and large intestine (Basic Protocol). describes a strategy to enrich the hematopoietic cell fraction in the sample by Percoll gradient centrifugation, and describes preparation of single cell suspensions from specific tissue layers of the small intestine, such as the epithelium, villi mucosa, submucosa, and muscularis externa. Finally, Support Protocol explains how to purify specific intestinal DC and macrophage subsets by flow‐cytometry‐based cell sorting. © 2016 by John Wiley & Sons, Inc.

Keywords: flow cytometry; mouse intestinal dendritic cells; macrophage subsets

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Analysis of Intestinal Mononuclear Phagocyte (MP) Subsets by Flow Cytometry
  • Alternate Protocol 1: Enrichment of Intestinal Hematopoietic Cells by Percoll Gradient Centrifugation
  • Alternate Protocol 2: Preparation of Single Cell Suspensions from The Small Intestinal Layers
  • Support Protocol 1: Purification of Intestinal Mononuclear Phagocyte (MP) Subsets by Cell Sorting
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Analysis of Intestinal Mononuclear Phagocyte (MP) Subsets by Flow Cytometry

  Materials
  • Mice (e.g., strain C57Bl/6), 6 to 8 weeks old
  • 70% alcohol
  • Complete HBSS, 4°C (see recipe)
  • Complete HBSS with dithiothreitol (DTT), 4°C (see recipe)
  • Complete HBSS with EDTA, 4°C (see recipe)
  • Complete RPMI medium with collagenase type IV, 4°C (see recipe)
  • Fluorescence‐activated cell sorting (FACS) buffer, 4°C (see recipe)
  • Trypan blue solution
  • Directly conjugated anti‐mouse monoclonal antibodies:
    • CD45 (clone 30‐F11)
    • MHC Class II (I‐A/I‐E, clone M5/114.15.2)
    • CD11c (clone N418)
    • CD11b (clone M1/70)
    • CD103 (clone 2E7)
    • CD16/CD32 (clone 93)
  • 4′,6‐Diamidino‐2‐phenylindole (DAPI) solution (see recipe)
  • Dissection tools (microdissecting scissors, curved Vannas spring scissors, two pairs of standard 1.3‐mm tip curved forceps, Dumont forceps)
  • 50‐ml Falcon conical tubes
  • 5‐ml FACS polystyrene round bottom tubes
  • 6‐well cell culture plates
  • 5‐ml syringe with 19‐G needle
  • 40‐ and 70‐μm nylon mesh cell strainers
  • 40‐μm nylon mesh, cut in 2‐cm square pieces
  • Hemocytometer
  • Inverted tissue culture microscope
  • Thermostat‐controlled bench‐top orbital shaking incubator, 37°C
  • Centrifuge with rotor adapters for 15‐ and 50‐ml Falcon tubes and 5‐ml FACS tubes
  • Humidified cell culture incubator, 5% CO 2, 37 °C
  • Becton Dickinson 15‐color LSR II Special Order flow cytometer running FACSDiva 6 software (BD)
  • FlowJo X software (Tree Star)
  • Additional reagents and equipment for trypan blue cell viability test ( appendix 3B)
  • Additional reagents and equipment for euthanizing mice (unit 1.8, Donovan and Brown, )
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Figures

Videos

Literature Cited

Literature Cited
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