Performing Bronchoalveolar Lavage in the Mouse

François Daubeuf1, Nelly Frossard1

1 UMR 7200 CNRS—Université de Strasbourg, Laboratoire d'Innovation Thérapeutique, Faculté de Pharmacie, Illkirch, France
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo110201
Online Posting Date:  June, 2012
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Abstract

Bronchoalveolar lavage (BAL) is a simple technique commonly used in humans to sample the contents of the epithelial lining fluid and determine the cellular and molecular composition of the pulmonary airways. In murine models, BAL makes it possible to sample immunological and inflammatory cell populations; it is indispensable for studying cell influx in disease models of the airways such as asthma and COPD. Cell counts can be combined with methods such as ELISA, immunoblot, immunohistochemistry, quantitative polymerase chain reaction, and HPLC to assess such inflammatory components as cytokines, growth factors, analytes, and receptors expressed at the cell membrane. Performing BAL in a reproducible manner is a hallmark of airway research in the mouse. Several procedures may be implemented. This unit describes a basic, rapid, inexpensive, and highly reproducible procedure to collect BAL fluid and cells that can be counted efficiently and reproducibly. Curr. Protoc. Mouse Biol. 2:167‐175 © 2012 by John Wiley & Sons, Inc.

Keywords: bronchoalveolar lavage; inflammation; airways; lung; asthma; COPD

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Bronchoalveolar Lavage
  • Basic Protocol 2: Cell Counts
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Bronchoalveolar Lavage

  Materials
  • Anesthetic (Ketamine 50 mg/kg‐Xylazine 3 mg/kg)
  • Sterile saline with EDTA added (2.6 mM) on ice
  • 1‐ml sterile syringes
  • 21‐ and 25‐G sterile needles
  • 23‐G sterile needles, optional (if sampling blood)
  • Scissors
  • Cotton thread no. 40
  • 21‐G lavage tubing, carefully placed over a 21‐G needle
  • 5‐ml polypropylene tubes
  • Centrifuge

Basic Protocol 2: Cell Counts

  Materials
  • BAL (see protocol 1)
  • Sterile deionized H 2O (H 2Od)
  • Sterile potassium chloride solution (0.6 M)
  • Sterile saline (0.9% NaCl) with EDTA (2.6 mM) on ice
  • Diff‐Quick staining kit (Hemacolor; Merck, cat. no. 1.11661.0001) containing:
    • Diff‐Quick fixative
    • Solution I
    • Solution II
  • Reagent HMS‐A (Home‐Made stain A, methanol 100%)
  • Reagent HMS‐B (Eosin 0.075%, H 2Od)
  • Reagent HMS‐C (methylene blue 0.06%, toluidine blue 0.04%, H 2Od)
  • Centrifuge
  • Hemacytometer (Neubauer or other)
  • Cytofunnel (Shandon; Thermo Scientific, cat. no. 5991040)
  • Shandon filter cards (Shandon; Thermo Scientific, cat. no. 5991022)
  • Superfrost slides (MENZEL‐GLÄSER; Thermo Scientific, cat. no. LCSF)
  • Cytospin (Shandon; Thermo Scientific, cat. no. A7830002)
  • Conventional brightfield microscope
  • Automated stainer Shandon Varistain XY (with low agitation)
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Figures

Videos

Literature Cited

Literature Cited
   Andreasen, C.B. 2003. Bronchoalveolar lavage. Vet. Clin. Small Anim. 33:69‐88.
   Blé, F.‐X, Cannet, C., Zurbruegg, S., Karmouty Quintana, H., Frossard, N., Trifilieff, A., and Beckmann, N. 2008. Allergen‐induced lung inflammation in actively sensitized mice assessed by MRI. Radiology 248:834‐843.
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   Henderson, A.J.W. 1994. Bronchoalveolar lavage. Arch. Dis. Childhood 70:167‐169.
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   Reber, L.L., Daubeuf, F., Plantinga, M., De Cauwer, L., Gerlo, S., Waelput, W., Van Calenbergh, S., Tavernier, J., Haegeman, G., Lambrecht, B.N., Frossard, N., and de Bosscher, K. 2012. A fully dissociated glucocorticoid receptor modulator reduces airway hyperresponsiveness and inflammation in a murine model of asthma. J. Immunol. 188:3478‐3487.
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