Mouse Eosinophils: Identification, Isolation, and Functional Analysis

Hadar Reichman1, Perri Rozenberg1, Ariel Munitz1

1 Department of Clinical Microbiology and Immunology, The Sackler School of Medicine, Tel‐Aviv University, Ramat Aviv
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.43
DOI:  10.1002/cpim.35
Online Posting Date:  November, 2017
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Abstract

Eosinophils are bone marrow–derived cells that differentiate in the bone marrow and migrate into the peripheral blood primarily under the regulation of interleukin (IL)–5. Eosinophil levels in the blood are relatively low. However, under steady‐state conditions and in settings of allergic inflammation, parasite infections, or even cancer, they migrate and mainly reside in mucosal tissues where they have key effector and immune‐modulating functions. Functional studies using eosinophils are not simple, since these cells are terminally differentiated and rapidly die in vitro. Thus, establishing simple methods to characterize, obtain, and functionally assess eosinophil activities is important. In this unit, we describe methodology for identifying tissue eosinophils by flow cytometry. In addition, we provide detailed methods for isolating eosinophils and for differentiating them from bone marrow cells for further functional studies. © 2017 by John Wiley & Sons, Inc.

Keywords: eosinophils; inflammation; chemotaxis; flow cytometry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Identification of Tissue Eosinophils by Flow Cytometry
  • Support Protocol 1: Enzymatic Digestion of Mouse Adipose Tissue
  • Support Protocol 2: Enzymatic Digestion of Mouse Colon/Ileum Tissue
  • Basic Protocol 2: Isolation of Eosinophils From IL‐5 Transgenic (Il5Tg) Mice
  • Basic Protocol 3: In Vitro Generation of Eosinophils Using Bone Marrow‐Derived Cultures
  • Support Protocol 3: Isolation of Lineage‐Negative Cells From Bone Marrow
  • Support Protocol 4: Identification of BM‐Derived Eosinophils in Culture
  • Basic Protocol 4: Functional Activation of Eosinophils Using Chemokines: Assessment of Calcium Flux
  • Basic Protocol 5: Functional Activation of Eosinophils Using Chemokines: Assessment of Actin Polymerization
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Identification of Tissue Eosinophils by Flow Cytometry

  Materials
  • Tissue sample as single‐cell suspension in HBA buffer (see protocol 2)
  • HBA buffer (see recipe)
  • Antibodies: the following is an example of a possible antibody combination; antibodies should be chosen according to the flow machine used and used per manufacturer's instructions:
    • APC‐anti–mouse CD45 (eBioscience, cat. no. 17‐0451‐82)
    • PerCP‐Cy5.5‐anti–mouse CD11b (eBioscience, cat. no. 45‐0112‐80)
    • PE‐anti–mouse Siglec‐F (BD Bioscience cat. no. 552126)
  • 4′,6‐diamidino‐2‐phenylindole (DAPI; Sigma, cat. no. D9564)
NOTE: Any combination of the aforementioned antibodies will be sufficient depending on the available filters that accompany the respective flow cytometer.NOTE: This antibody “cocktail” can be easily integrated with additional cell surface markers such as anti‐Ly6C, anti‐Ly6G, anti‐CD11c, and anti‐MHC‐II, which will provide identification of a larger myeloid panel.

Support Protocol 1: Enzymatic Digestion of Mouse Adipose Tissue

  Materials
  • Mouse
  • Enzyme mix (see recipe)
  • RBC lysis buffer (see recipe)
  • HBA buffer (see recipe)
  • Clinical rotator
  • 70‐µm‐mesh cell strainer
  • Refrigerated centrifuge
  • Additional reagents and equipment for dissection of mice (Antal, Muller, Wendling, Hérault, & Mark, ; Treuting & Snyder, ) and flow cytometry (Chapter 5)

Support Protocol 2: Enzymatic Digestion of Mouse Colon/Ileum Tissue

  Materials
  • Dulbecco's phosphate‐buffered saline (PBS; Sigma, cat. no. D8537)
  • Solution 1 (see recipe)
  • Solution 2 (see recipe)
  • HBA buffer (see recipe)
  • 50‐ml conical tubes (e.g., Corning Falcon)
  • 70‐ to 100‐μm mesh cell strainer
  • Clinical rotator
  • Refrigerated centrifuge
  • Additional reagents and equipment for dissection of mice (Antal et al., ; Treuting & Snyder, ) and flow cytometry (Chapter 5)

Basic Protocol 2: Isolation of Eosinophils From IL‐5 Transgenic (Il5Tg) Mice

  Materials
  • Cells from peritoneal lavage (Munitz et al., ; Zhang, Goncalves, & Mosser, )
  • MACS buffer (see recipe)
  • Biotinylated antibodies (Miltenyi Biotec):
    • For negative selection: CD45R (cat. no. 130‐101‐928), CD90.2 (cat. no. 130‐101‐908)
    • For positive selection: Siglec‐F (cat. no. 130‐101‐861)
  • Anti‐biotin microbeads (Miltenyi Biotec, cat. no. 130‐090‐485)
  • Dynabeads (Thermo Fisher Scientific): anti‐T (Thy1.2, cat. no. 114.43D), anti‐B (B220, cat. no. 114.41D)
  • Isolation buffer (see recipe in reagents and solutions)
  • Refrigerated centrifuge
  • MS columns (Miltenyi Biotec, cat. no. 130‐042‐201)
  • Magnetic cell separator suitable for MS columns (multiple products available)
  • Platform rotator providing gentle tilting and rotation
  • Additional reagents and equipment for counting cells ( appendix 3A; Strober, )

Basic Protocol 3: In Vitro Generation of Eosinophils Using Bone Marrow‐Derived Cultures

  Materials
  • Mouse, 1 to 12 months old
  • Hank's Balanced Salt Solution (HBSS; Biological Industries, cat. no. 02‐017‐1A)
  • 70% ethanol
  • Red blood cell lysis buffer (Hybri‐Max™; Sigma, cat. no. 7757)
  • Histopaque®‐1083 (Sigma, cat. no. 10831)
  • Eosinophil growth medium (see recipe)
  • 15‐ml conical polypropylene tubes (e.g., Corning Falcon)
  • Mortar (Sigma, cat. no. Z247480)
  • Pestle (Sigma, cat. no. Z247529)
  • 70‐µm cell strainer
  • Refrigerated centrifuge
  • 50‐ml polypropylene tubes (e.g., Corning Falcon)
  • Additional reagents and equipment for dissection of mice (Antal et al., ; Treuting & Snyder, ) and counting cells ( appendix 3A; Strober, )

Support Protocol 3: Isolation of Lineage‐Negative Cells From Bone Marrow

  Materials
  • Cells ( protocol 5)
  • Eosinophil growth medium (see recipe)
  • MACS buffer (see recipe), sterile
  • Lineage Cell Depletion Kit, mouse (Miltenyi Biotec, cat. no. 130‐090‐858)
  • Recombinant Murine Flt3‐Ligand (Peprotech Asia, cat. no. 250‐31L)
  • Recombinant Murine SCF (Peprotech Asia, cat. no. 250‐03)
  • Recombinant Murine IL‐5 (Peprotech Asia, cat. no. 215‐15)
  • Refrigerated centrifuge
  • MS columns (Miltenyi Biotec, cat. no. 130‐042‐201)
  • 15‐ml conical tubes (e.g., Corning Falcon)
  • 24‐well tissue culture plate
  • Additional reagents and equipment for counting cells ( appendix 3A; Strober, ) and flow cytometry (Chapter 5)

Support Protocol 4: Identification of BM‐Derived Eosinophils in Culture

  Materials
  • Eosinophils in 24‐well plate ( protocol 6)
  • HBA buffer (see recipe)
  • Anti‐mouse CCR3 FITC (R&D Systems, cat. no. FAB729F)
  • Anti‐mouse Siglec‐F PE (BD Bioscience, cat. no. 552126)
  • Additional reagents and equipment for counting cells ( appendix 3A; Strober, ) and flow cytometry (Chapter 5)
NOTE: The aforementioned antibodies are an example for antibody combination. The antibodies should be chosen according to the specific abilities of the flow cytometer used.

Basic Protocol 4: Functional Activation of Eosinophils Using Chemokines: Assessment of Calcium Flux

  Materials
  • Mouse
  • Hank's Balanced Salt Solution (HBSS; Biological Industries, cat. no. 02‐017‐1A)
  • Red Blood Cell Lysing Buffer (Hybri‐Max™; Sigma, cat. no. 7757)
  • HBA buffer (see recipe)
  • Anti‐mouse CCR3 Alexa Fluor 700 (R&D Systems, cat. no. FAB729N)
  • Anti‐mouse Siglec‐F PE (BD Bioscience, cat. no. 552126)
  • Anti‐mouse CD45 APC (eBioscience, cat. no. 17‐0451‐82)
  • DPBS+/+: Dulbecco's phosphate‐buffered saline with MgCl 2 and CaCl 2 (Sigma, cat. no. D8662)
  • Fluo‐4‐AM, cell permeant (Invitrogen, cat. no. F14201)
  • 15‐ml conical polypropylene tubes
  • Mortar (Sigma, cat. no. Z247480)
  • Pestle (Sigma, cat. no. Z247529)
  • 70‐µm cell strainer
  • Refrigerated centrifuge
  • Flow cytometer tubes
  • Additional reagents and equipment for dissection of mice (Antal et al., ; Treuting & Snyder, ), counting cells ( appendix 3A; Strober, ), and flow cytometry (Chapter 5)

Basic Protocol 5: Functional Activation of Eosinophils Using Chemokines: Assessment of Actin Polymerization

  Materials
  • Mouse
  • Hank's Balanced Salt Solution (HBSS; Biological Industries, cat. no. 02‐017‐1A)
  • Red Blood Cell Lysing Buffer (Hybri‐Max™; Sigma, cat. no. 7757)
  • Fetal bovine serum (FBS; Biological Industries, cat. no. 04‐001‐1A)
  • Recombinant murine eotaxin‐1 (CCL11; Peprotech Asia, cat. no. 250‐01)
  • Recombinant murine eotaxin‐2 (CCL24; Peprotech Asia, cat. no. 250‐22)
  • 16% (w/v) formaldehyde, methanol‐free (Thermo Scientific, cat. no. 28906)
  • Anti–mouse CCR3 Alexa Fluor 700 (R&D Systems, cat. no. FAB729N)
  • Anti–mouse Siglec‐F PE (BD Bioscience, cat. no. 552126)
  • Anti–mouse CD45 APC (eBioscience, cat. no. 17‐0451‐82)
  • FITC‐phalloidin (Sigma, cat. no. P5282)
  • Saponin, 1% in H 2O (Sigma, cat. no. S7900)
  • Dulbecco's phosphate‐buffered saline without Ca or Mg (DPBS; Sigma, cat. no. D8537)
  • Bovine serum albumin (BSA; Amresco, cat. no. 0332‐TAM)
  • 15‐ml conical tubes (e.g., Corning Falcon)
  • Mortar (Sigma, cat. no. Z247480)
  • Pestle (Sigma, cat. no. Z247529)
  • 70‐µm cell strainer
  • 50‐ml conical tubes (e.g., Corning Falcon)
  • Refrigerated centrifuge
  • 96‐well U‐bottom plate
  • Additional reagents and equipment for dissection of mice (Antal et al., ; Treuting & Snyder, ), counting cells ( appendix 3A; Strober, ), and flow cytometry (Chapter 5)
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Figures

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Literature Cited

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