A Fast, Easy, and Customizable Eight‐Color Flow Cytometric Method for Analysis of the Cellular Content of Bronchoalveolar Lavage Fluid in the Mouse

François Daubeuf1, Julien Becker2, Juan Antonio Aguilar‐Pimentel3, Claudine Ebel4, Martin Hrabě de Angelis3, Yann Hérault5, Nelly Frossard1

1 Centre National de la Recherche Scientifique, UMR 7200, Illkirch Cedex, 2 CELPHEDIA, PHENOMIN, Institut Clinique de la Souris (ICS), Illkirch‐Graffenstaden, 3 German Mouse Clinic, Helmholtz Zentrum München – Deutsches Forschungszentrum für Gesundheit und Umwelt, Neuherberg, 4 Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, 5 Université de Strasbourg, Illkirch
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/cpmo.26
Online Posting Date:  June, 2017
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The cell composition of bronchoalveolar lavage fluid (BAL) is an important indicator of airway inflammation. It is commonly determined by cytocentrifuging leukocytes on slides, then staining, identifying, and counting them as eosinophils, neutrophils, macrophages, or lymphocytes according to morphological criteria under light microscopy, where it is not always easy to distinguish macrophages from lymphocytes. We describe here a one‐step, easy‐to‐use, and easy‐to‐customize 8‐color flow cytometric method for performing differential cell count and comparing it to morphological counts on stained cytospins. This method identifies BAL cells by a simultaneous one‐step immunolabeling procedure using antibodies to identify T cells, B cells, neutrophils, eosinophils, and macrophages. Morphological analysis of flow‐sorted cell subsets is used to validate this protocol. An important advantage of this basic flow cytometry protocol is the ability to customize it by the addition of antibodies to study receptor expression at leukocyte cell surfaces and identify subclasses of inflammatory cells as needed. © 2017 by John Wiley & Sons, Inc.

Keywords: flow cytometry; bronchoalveolar lavage; inflammation; asthma; macrophages

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Table of Contents

  • Introduction
  • Basic Protocol 1: Flow Cytometry Procedure and Analysis
  • Support Protocol 1: Total Cell Counts
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Flow Cytometry Procedure and Analysis

  • Bronchoalveolar lavage (BAL) fluid from mouse
  • Phosphate buffered saline containing 3 mM EDTA (PBS‐EDTA), kept on ice
  • FC Block solution (to block non‐specific FcγII/IIIR antibody binding and reduce background staining; BD Bioscience, cat. no. 553142)
  • 4′,6‐diamidino‐2‐phenylindole (DAPI; fluorescent dye for exclusion of nonviable cells on flow cytometric analysis; BD Bioscience, cat. no. 564907)
  • Anti‐mouse antibodies (see Table 16.2.100)
    • CD45‐AlexaFluor700
    • CD11b‐APC Alexa750
    • Ly‐6G (Gr‐1)‐PEeFluor610
    • CD11c‐FITC
    • I‐A/I‐E‐PerCP‐Cy5.5
    • CD3‐BV605
    • CD19‐BV650
  • 1.5‐ml protein Lobind microtubes (Eppendorf, cat. no. 0030108116)
  • MixMate vortex (Eppendorf)
  • Sterile tips
  • Precision pipets (2.5, 20, 200, and 1000 μl)
  • 8‐color or more flow cytometer (LSR II or FACS Aria II, BD Bioscience)
  • FlowJo software
Table 6.2.1   MaterialsMonoclonal Antibodies for Flow Cytometry Analysis of Leukocytes in BAL Fluid in the Mouse a

Antibody Fluorochrome Supplier Catalog number Concentration Laser Filter
General panel
Live‐Dead DAPI BD Bioscience 564907 10 µg/ml Violet (405 nm) 450/50
CD45 AlexaFluor700 BioLegend 103128 1.7 µg/ml Red (633 nm) 730/45
CD11b APC Alexa750 BD Bioscience 557657 0.8 µg/ml Red (633 nm) 780/60
CD11c FITC BD Bioscience 557400 1.5 µg/ml Blue (488 nm) 530/30
CD3 BV605 BD Bioscience 564009 0.2 µg/ml Violet (405 nm) 605/12
CD19 BV650 BD Bioscience 563235 0.2 µg/ml Violet (405 nm) 655/8
I‐A/I‐E‐(MHC‐II) PerCP‐Cy5.5 BioLegend 107626 0.2 µg/ml Blue (488 nm) 695/40
Ly‐6 G (GR‐1) Pe‐eFluor 610 eBioscience 61‐5931 0.4 µg/ml Blue (488 nm) 610/20
Supplementary antibodies used for results presented in Figure 3
F4/80 BV510 BioLegend 123135 1 µg/ml Violet (405 nm) 525/25
CD206 (MMR) PE‐CY7 BioLegend 141719 1 µg/ml Blue (488 nm) 780/60
CD197 (CCR7) PerCP‐Cy5.5 eBioscience 45‐1971 1 µg/ml Blue (488 nm) 695/40
CXCR4 (CD184) APC eBioscience 17‐9991 1.5 µg/ml Red (633 nm) 660/20
CD4 APC BD Bioscience 561091 0.5 µg/ml Red (633 nm) 660/20

 aThe “General panel” of antibodies refers to results illustrated in Figure 1, and the “Supplementary antibodies” give results as presented in Figure 3.

Support Protocol 1: Total Cell Counts

  • Bronchoalveolar lavage (BAL) fluid from mouse
  • Phosphate buffered saline containing 3 mM EDTA (PBS‐EDTA), kept on ice
  • PBS‐EDTA containing 1% BSA (PBS‐BSA), kept on ice
  • Anti‐mouse antibody, CD45‐PE (Abcam, ab25603)
  • Conventional bright field microscope
  • Hemocytometer (e.g., Neubauer, Malassez)
  • Muse cytometer (Millipore)
  • Precision pipets (2.5, 20, 200, and 1000 μl)
  • Sterile tips
  • 1.5‐ml microtube
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Literature Cited

Literature Cited
  Abboud, D., Daubeuf, F., Do, Q. T., Utard, V., Villa, P., Haiech, J., … Frossard, N. (2015). A strategy to discover decoy chemokine ligands with an anti‐inflammatory activity. Scientific Reports, 5, 14746. doi: 10.1038/srep14746.
  Andreasen, C. B. (2003). Bronchoalveolar lavage. Veterinary Clinics of North America: Small Animal Practice, 33, 69–88. doi: 10.1016/S0195‐5616(02)00056‐6.
  Blé, F.‐X., Cannet, C., Zurbruegg, S., Karmouty‐Quintana, H., Frossard, N., Trifilieff, A., & Beckmann, N. (2008). Allergen‐induced lung inflammation in actively sensitized mice assessed by MRI. Radiology, 248(3): 834–843. doi: 10.1148/radiol.2482071452.
  Blé, F.‐X., Cannet, C., Zurbruegg, S., Gérard, C., Frossard, N., Beckmann, N., & Trifilieff, A. (2009). Activation of the lung S1P1 receptor reduces allergen‐induced plasma leakage in mouse. British Journal of Pharmacology, 158, 1295–1301. doi: 10.1111/j.1476‐5381.2009.00391.x.
  Crestani, B., Marchand‐Adam, S., Quesnel, C., Plantier, L., Borensztajn, K., Marchal, J., … Dehoux, M. (2012). Hepatocyte growth factor and lung fibrosis. Proceedings of the American Thoracic Society, 9, 158–163. doi: 10.1513/pats.201202‐018AW.
  Daubeuf, F., & Frossard, N. (2012). Performing bronchoalveolar lavage in the mouse. Current Protocols in Mouse Biology, 2, 167–175. doi: 10.1002/9780470942390.mo110201.
  Daubeuf, F., & Frossard, N. (2013). Acute ovalbumin asthma model in the mouse. Current Protocols in Mouse Biology, 3, 31–37. doi: 10.1002/9780470942390.mo120202.
  Delayre‐Orthez, C., Becker, J., Auwerx, J., Frossard, N., & Pons, F. (2008). Suppression of allergen‐induced airway inflammation and immune response by the peroxisome proliferator‐activated receptor‐alpha agonist fenofibrate. European Journal of Pharmacology, 581, 177–184. doi: 10.1016/j.ejphar.2007.11.040.
  Delayre‐Orthez, C., Becker, J., De Blay, F., Frossard, N., & Pons, F. (2004). Dose‐dependent effects of endotoxins on allergen sensitization and challenge in the mouse. Clinical and Experimental Allergy, 34, 1789–1795. doi: 10.1111/j.1365‐2222.2004.02082.x.
  Delayre‐Orthez, C., Becker, J., de Blay, F., Frossard, N., & Pons, F. (2005a). Exposure to endotoxins during sensitization prevents further endotoxin‐induced exacerbation of airway inflammation in a mouse model of allergic asthma. International Archives of Allergy and Immunology, 138, 298–304. doi: 10.1159/000088867.
  Delayre‐Orthez, C., Becker, J., Guenon, I., Lagente, V., Auwerx, J., Frossard, N., & Pons, F. (2005b). PPARalpha downregulates airway inflammation induced by lipopolysaccharide in the mouse. Respiratory Research, 6, 91. doi: 10.1186/1465‐9921‐6‐91.
  Deschamps, K., Cromlish, W., Weicker, S., Lamontagne, S., Huszar, S. L., Gauthier, J. Y., … Tan, C. M. (2011). Genetic and pharmacological evaluation of Cathepsin S in a mouse model of asthma. American Journal of Respiratory Cell and Molecular Biology, 45, 81–87. doi: 10.1165/rcmb.2009‐0392OC.
  Fuchs, H., Gailus‐Durner, V., Adler, T., Aguilar‐Pimentel, J. A., Becker, L., Calzada‐Wack, J., … Hrabě de Angelis, M. (2011). Mouse phenotyping. Methods, 53, 120–135. doi: 10.1016/j.ymeth.2010.08.006.
  Gasparik, V., Daubeuf, F., Hachet‐Haas, M., Rohmer, F., Gizzi, P., Haiech, J., … Frossard, N. (2012). Prodrugs of a CXC Chemokine‐12 (CXCL12) neutraligand prevent inflammatory reactions in an asthma model in vivo. ACS Medicinal Chemistry Letters, 3, 10–14. doi: 10.1021/ml200017d.
  Gregory, L. G., & Lloyd, C. M. (2011). Orchestrating house dust mite‐associated allergy in the lung. Trends in Immunology, 32, 402–411. doi: 10.1016/j.it.2011.06.006.
  Hachet‐Haas, M., Balabanian, K., Rohmer, F., Pons, F., Franchet, C., Lecat, S., … Galzi J.‐L. (2008). Small neutralizing molecules to inhibit actions of the chemokine CXCL12. The Journal of Biological Chemistry, 283, 23189–23199. doi: 10.1074/jbc.M803947200.
  Henderson, A. J. W. (1994). Bronchoalveolar lavage. Archives of Disease in Childhood, 70, 167–169. doi: 10.1136/adc.70.3.167.
  Horsch, M., Aguilar‐Pimentel, J. A., Bonisch, C., Come, C., Kolster‐Fog, C., Jensen, K. T., … Beckers, J. (2015). Cox4i2, Ifit2, and Prdm11 mutant mice: Effective selection of genes predisposing to an altered airway inflammatory response from a large compendium of mutant mouse lines. PloS One, 10, e0134503. doi: 10.1371/journal.pone.0134503.
  Hunninghake, G. W., Gadek, J. E., Kawanami, O., Ferrans, V. J., & Crystal, R. G. (1979). Inflammatory and immune processes in the human lung in health and disease: Evaluation by bronchoalveolar lavage. The American Journal of Pathology, 97, 149–206.
  Kips, J. C., Anderson, G. P., Fredberg, J. J., Herz, U., Inman, M. D., Jordana, M., … Chung, K. F. (2003). Murine models of asthma. The European Respiratory Journal, 22, 374–382. doi: 10.1183/09031936.03.00026403.
  Kumar, R. K., Herbert, C., & Foster, P. S. (2008). The “classical” ovalbumin challenge model of asthma in mice. Current Drug Targets, 9, 485–494. doi: 10.2174/138945008784533561.
  Mathers, R. A., Evans, G. O., Bleby, J., & Tornow, T. (2007). Total and differential leucocyte counts in rat and mouse bronchoalveolar lavage fluids using the Sysmex XT‐2000iV. Comparative Clinical Pathology, 16, 29–39. doi: 10.1007/s00580‐006‐0655‐x.
  Mouratis, M. A. (2011). Modeling pulmonary fibrosis with bleomycin. Current Opinion in Pulmonary Medicine, 17, 355–361. doi: 10.1097/MCP.0b013e328349ac2b.
  Nials, A. T., & Uddin, S. (2008). Mouse models of allergic asthma: Acute and chronic allergen challenge. Disease Models & Mechanisms, 1, 213–220. doi: 10.1242/dmm.000323.
  Reber, L. L., Daubeuf, F., Pejler, G., Abrink, M., & Frossard, N. (2014). Mast cells contribute to bleomycin‐induced lung inflammation and injury in mice through a chymase/mast cell protease 4‐dependent mechanism. Journal of Immunology, 192, 1847–1854. doi: 10.4049/jimmunol.1300875.
  Shin, Y. S., Takeda, K., & Gelfand, E. W. (2009). Understanding asthma using animal models. Allergy, Asthma & Immunology Research, 1, 10–18. doi: 10.4168/aair.2009.1.1.10.
  Van Rijt, L. S., Kuipers, H., Vos, N., Hijdra, D., Hoogsteden, H. C., & Lambrecht, B. N. (2004). A rapid flow cytometric method for determining the cellular composition of bronchoalveolar lavage fluid cells in mouse models of asthma. Journal of Immunological Methods, 288, 111–221. doi: 10.1016/j.jim.2004.03.004.
  Vodovotz, Y., Chow, C. C., Bartels, J., Lagoa, C., Prince, J. M., Levy, R. M., … Fink, M. P. (2006). In silico models of acute inflammation in animals. Shock, 26, 235–244. doi: 10.1097/01.shk.0000225413.13866.fo.
  Zhang, Z., Hener, P., Frossard, N., Kato, S., Metzger, D., Li, M., & Chambon, P. (2009). Thymic stromal lymphopoietin overproduced by keratinocytes in mouse skin aggravates experimental asthma. Proceedings of the National Academy of Sciences of the U.S.A., 106(5), 1536–1541. doi: 10.1073/pnas.0812668106.
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