Generation and Characterization of Mouse Basophils from Bone Marrow and Purification of Basophils from Spleen

Tomohiro Yoshimoto1, Kenji Nakanishi2

1 Laboratory of Allergic Diseases, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Hyogo, Japan, 2 Department of Immunology and Medical Zoology, Hyogo College of Medicine, Hyogo, Japan
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.24
DOI:  10.1002/0471142735.im0324s98
Online Posting Date:  August, 2012
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Abstract

Basophils are rare circulating granulocytes that originate from progenitor cells in the bone marrow and have been considered important effector cells in IgE‐mediated allergic inflammation. Basophils constitute <1% of blood leukocytes and are usually absent or present only in small numbers in tissues. They may, however, be recruited to inflammatory sites when an antigen is present and contribute immediately to hypersensitivity reactions. Basophils can therefore serve as primary effector cells in allergic disorders. Despite a large pool of experimental evidence that has led to the discovery of these functional attributes of basophils, many questions regarding their contribution to these immune responses remain unanswered. This is due, in part, to the lack of methods for generation and purification of basophils and the lack of animal models appropriate for their functional analysis. Recent studies, however, have revealed a role for basophils as antigen‐presenting cells that preferentially induce Th2 cells in response to complexes of antigen plus antigen‐specific IgE, to protease allergens, or to helminth parasites in vitro and in vivo through the production of “early IL‐4” and the presentation to CD4+ T cells of complexes of peptide plus MHC class II molecules. These findings have uncovered previously unknown functional characteristics of basophils. Knowledge of these and other functional properties of basophils may translate into the design of novel therapeutic strategies for Th2‐IgE‐mediated diseases, such as bronchial asthma. In this unit, protocols that will enable the study of mouse basophils are described. Curr. Protoc. Immunol. 98:3.24.1‐3.24.16. © 2012 by John Wiley & Sons, Inc.

Keywords: mouse basophils; bone marrow; spleen; antigen‐presenting cells; cytokine; FcɛRI

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Generation and Isolation of Mouse Bone Marrow–Derived Basophils
  • Basic Protocol 2: Purification of Basophils from Spleen
  • Support Protocol 1: In Vivo Treatment of Naïve Mice with IL‐3 to Enhance Basophil Recovery
  • Basic Protocol 3: Surface Marker Characterization of Mouse Bone Marrow–Derived and Splenic Basophils
  • Basic Protocol 4: Characterization of FACS‐Sorted Basophils by Measuring Cytokine and Chemokine Production
  • Alternate Protocol 1: Characterization of Bone Marrow–Derived and FACS‐Sorted Basophils by Antigen‐Presenting Function
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Generation and Isolation of Mouse Bone Marrow–Derived Basophils

  Materials
  • Mice (8 to 12 months old, male)
  • RPMI 1640, ice cold
  • Ammonium chloride solution
  • Phosphate‐buffered saline (PBS; appendix 2A), ice cold
  • Supplemented RPMI 1640 culture medium (see recipe)
  • Mouse IL‐3 (e.g., R&D Systems)
  • Staining buffer (see recipe): PBS containing 1% (w/v) FBS
  • Anti‐CD16/32
  • Staining reagents: biotin anti‐mouse FcɛRIα (clone: MARI) (eBioscience), PE‐anti‐mouse c‐Kit (CD117; clone 2BB), PE‐anti‐mouse CD49b (clone DX5) (BD Bioscience), anti‐FcγRII/III (eBioscience), and streptavidin‐APC (BioLegend)
  • Dissecting instruments: scissors and forceps
  • 100‐mm tissue culture dishes (e.g., Falcon)
  • 10‐ml syringes
  • 25‐G needles
  • 50‐ml polypropylene conical centrifuge tubes (Falcon, no. 2070), sterile
  • Refrigerated centrifuge
  • Cell strainer (BD Falcon, cat. no. 352350)
  • 5‐ml polystyrene round‐bottom tubes (Falcon, no. 2058)
  • Fluorescence cell sorter (FACS Aria; BD Biosciences)
  • Additional reagents and equipment for euthanasia (unit 1.8), counting cells using a hemacytometer ( appendix 3A), and toluidine blue staining of basophils (unit 7.25)
NOTE: All procedures, including bone removal and femur lavage, are performed in a laminar flow hood. All reagents are sterilized by filtration through a 0.22‐µm filter, and all surgical equipment is sterilized by immersion in 70% ethanol prior to use.NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator.

Basic Protocol 2: Purification of Basophils from Spleen

  Materials
  • Mice (8 to 12 months old)
  • Ammonium chloride solution (unit 2.5)
  • PBS ( appendix 2A)
  • Mouse CD90.1 and B220 MicroBeads (Miltenyi Biotec)
  • Magnetic‐activated cell‐sorting (MACS) wash solution (see recipe), ice cold
  • Staining buffer (see recipe): PBS containing 1% (w/v) FBS, 4°C
  • Supplemented RPMI 1640 culture medium (see recipe)
  • Dissecting instruments: scissors and forceps
  • 100‐mm tissue‐culture dishes (e.g., Falcon)
  • 50‐ml polypropylene conical centrifuge tubes (Falcon, no. 2070)
  • Refrigerated centrifuge
  • AutoMACS Separator (Miltenyi Biotec)
  • 5‐ml polystyrene round‐bottom tube (Falcon, no. 2058)
  • Flow cytometer (Chapter 5; FACS Calibur; BD Biosciences)
  • Additional reagents and equipment for euthanasia (unit 1.8), counting cells using a hemacytometer ( appendix 3A), isolating basophils (see protocol 1), and toluidine blue staining of splenic cells and basophils (unit 7.25)

Support Protocol 1: In Vivo Treatment of Naïve Mice with IL‐3 to Enhance Basophil Recovery

  Materials
  • Mouse IL‐3 (e.g., R&D Systems)
  • Isoflurane
  • Mice (8 to 12 months old)
  • Micro‐osmotic pumps (ALZET; model no. 1002)
  • 1‐ml syringes
  • Blunt‐tipped 27‐G needles
  • Surgical instruments
  • Surgical staples for mice (e.g., Autoclip 9‐mm, Stoelting Co.)

Basic Protocol 3: Surface Marker Characterization of Mouse Bone Marrow–Derived and Splenic Basophils

  Materials
  • Sorted bone marrow–derived basophils (see protocol 1) or splenic basophils (see protocol 2)
  • Staining buffer (see recipe): PBS containing 0.1% (w/v) BSA, 4°C
  • FITC‐anti‐mouse I‐Ad, FITC‐anti‐mouse CD40, FITC‐anti‐mouse CD80, FITC‐anti‐mouse CD86, FITC‐anti‐mouse CD11c, and FITC‐anti‐mouse CD62L (BD Biosciences)
  • 96‐well flat‐bottom plate
  • Refrigerated centrifuge with plate rotor
  • Plate mixer
  • Flow cytometer (Chapter 5)

Basic Protocol 4: Characterization of FACS‐Sorted Basophils by Measuring Cytokine and Chemokine Production

  Materials
  • Sorted bone marrow–derived basophils (see protocol 1) or splenic basophils (see protocol 2)
  • Mouse IL‐2, IL‐3, IL‐4 (e.g., R&D Systems), mouse IL‐18 (MBL), and IL‐33 (e.g., R&D Systems)
  • Cytokine (IL‐4, IL‐6, or IL‐13) ELISA kits (e.g., R&D Systems) or Bio‐Plex Mouse Cytokine 23‐Plex panel (BioRad)
  • 96‐well plates

Alternate Protocol 1: Characterization of Bone Marrow–Derived and FACS‐Sorted Basophils by Antigen‐Presenting Function

  Additional Materials
  • Mice transgenic for αβ T‐cell receptor (TCR) recognizing OVA 323‐339 (DO11.10)
  • CD4+CD62L+T cell isolation kit II for mouse (Miltenyi Biotec, cat. no. 130‐093‐227), optional
  • Isolated basophils (see Basic Protocols protocol 11 and protocol 22)
  • Mouse IL‐2, IL‐3, IL‐4 (e.g., R&D Systems), mouse IL‐18 (MBL), and IL‐33 (e.g., R&D Systems)
  • OVA peptide (OVA 323‐339) (e.g., AnaSpec Inc.)
  • Supplemented RPMI 1640 culture medium (see recipe)
  • Phorbol 12‐myristate 13‐acetate (PMA) (Sigma)
  • Ionomycin (Sigma)
  • Brefeldin A (BD GolgiPlug)
  • Ficoll‐Paque Plus (e.g., GE healthcare), optional
  • PBS ( appendix 2A)
  • PE‐anti‐IL‐4, FITC‐anti‐IFN‐γ, and APC‐anti‐CD4 (BD Biosciences)
  • Fixation and permeabilization solution kit with BD GolgiPlug (BD Biosciences, cat. no. 555028) containing:
    • BD Cytofix/Cytoperm solution
    • BD Perm/Wash buffer
    • BD GolgiPlug
  • 48‐ and 96‐well tissue‐culture plates (e.g., Costar)
  • Refrigerated centrifuge with plate adapter
  • Plate mixer, 4°C
  • Flow cytometer (Chapter 5)
  • Additional reagents and equipment for intracellular staining of cytokines (see unit 6.24), preparation of conventional APCs (see unit 16.1), isolation of splenic T cells (see units 3.3 or 3.5)
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Figures

Videos

Literature Cited

Literature Cited
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