Isolation of Thymic Epithelial Cells and Analysis by Flow Cytometry

Reema Jain1, Daniel H.D. Gray1

1 Department of Medical Biology, The University of Melbourne, Parkville, Victoria
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.26
DOI:  10.1002/0471142735.im0326s107
Online Posting Date:  November, 2014
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Abstract

The epithelial cells of the thymus govern the differentiation of hematopoietic precursors into T cells, which are critical for acquired immunity. Thymic epithelial cells (TEC) provide molecular cues that direct precursor recruitment, commitment to the T cell lineage, thymocyte proliferation, and the processes of positive and negative selection. Despite the importance of TEC to the immune system, fundamental questions regarding their differentiation, turnover, and function throughout life remain unanswered. This knowledge gap is largely due to technical difficulties in isolating, quantifying, and purifying this rare cell type. Here, we describe methods for the enzymatic digestion of the thymus to obtain single‐cell suspensions of TEC, their analysis by flow cytometry, enrichment using magnetic beads, and purification for a variety of downstream applications. © 2014 by John Wiley & Sons, Inc.

Keywords: thymic epithelial cells; enzymatic digestion; flow cytometry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Thymic Epithelial Cells by Enzymatic Digestion
  • Alternate Protocol 1: Isolation of Thymic Epithelial Cells From a Single Thymus by Enzymatic Digestion
  • Support Protocol 1: Immunofluorescent Staining for Flow Cytometry
  • Basic Protocol 2: Enrichment and Purification of Thymic Epithelial Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Thymic Epithelial Cells by Enzymatic Digestion

  Materials
  • Two to ten mice ethically euthanized by CO 2 asphyxiation (6 weeks old)
  • RPMI‐1640, ice cold
  • Digestion buffers I and II (see reciperecipes), in 37°C water bath
  • Dissecting instruments: scissors, straight, and curved forceps
  • 100‐mm plastic petri dishes
  • 10‐ml polypropylene conical centrifuge tubes (Techno Plas, cat. no. P10316), sterile
  • 1‐ml wide‐bore pipet tips
  • 200‐μl pipet tips
  • 37°C water bath
  • 100‐μm cell strainer (BD Falcon, cat. no. 352360)
  • Casy Counter or hemacytometer for cell counts
NOTE: For downstream applications requiring culture of isolated TEC, all procedures are performed in a laminar flow hood. All reagents are sterilized by filtration through a 0.22‐μm filter and handled with aseptic technique. Dissection equipment is sterilized by immersion in 70% ethanol and dried prior to use.

Alternate Protocol 1: Isolation of Thymic Epithelial Cells From a Single Thymus by Enzymatic Digestion

  Materials
  • Thymocyte and fractions (see protocol 1 or protocol 2Alternate Protocol)
  • FACS buffer (see recipe), ice cold
  • Antibody Cocktail I (see recipe), ice cold
  • Foxp3 Staining Buffer Set (eBioscience, cat. no. 00‐5523) containing:
    • Fixation/permeabilization concentrate (eBiosciences, cat. no. 00‐5123)
    • Fixation/permeabilization diluent (eBiosciences, cat. no. 00‐5223)
    • 1× permeabilization buffer (eBiosciences, cat. no. 00‐8333)
  • AIRE (Clone 5H12; The Walter and Eliza Hall Institute Monoclonal Antibody Facility)
  • Propidium iodide (PI; Invitrogen, cat. no. P3566)
  • 96‐well U‐bottom plate (BD Falcon, cat, no, 353077)
  • Refrigerated centrifuge set at 4°C
  • Flow cytometer (e.g., LSR II, BD Biosciences)

Support Protocol 1: Immunofluorescent Staining for Flow Cytometry

  Materials
  • Pooled thymic fractions (“final fractions” from protocol 1)
  • FACS buffer (see recipe), ice cold
  • Anti‐mouse CD45 microbeads (Miltenyi Biotec, cat. no. 130052301)
  • Antibody cocktail I (see recipe)
  • Propidium iodide (PI; Invitrogen, cat. no. P3566)
  • Antibody cocktail II (see recipe)
  • 30% (v/v) FBS in RPMI (collection buffer)
  • 10‐ml polypropylene conical centrifuge tubes (Techno Plas, cat. no. P10316), sterile
  • Refrigerated centrifuge set at 4°C
  • AutoMACS machine (Miltenyi Biotec)
  • 100‐μm cell strainer
  • 5‐ml polypropylene round‐bottom tubes (BD Falcon, cat. no. 352063), sterile
  • Cell Sorter (e.g., Moflo, Beckman Counter or FACS ARIA, BD)
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Figures

Videos

Literature Cited

Literature Cited
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