Performing Bronchoalveolar Lavage in the Mouse
互联网
1624
- Abstract
- Table of Contents
- Materials
- Figures
- Literature Cited
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
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Basic Protocol 1: Bronchoalveolar Lavage
- Basic Protocol 2: Cell Counts
- Commentary
- Literature Cited
- Figures
- Tables
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online LibraryMaterials
Basic Protocol 1: Bronchoalveolar Lavage
Materials
Basic Protocol 2: Cell Counts
Materials
|
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online LibraryFigures
-
Figure 1. Evolution of total cell count per lavage in bronchoalveolar lavage (BAL). Total cells in BALF from control naive (Ctrl), ovalbumin‐sensitized and ‐challenged (OVA), and Escherichia coli lipopolysaccharide‐treated (LPS) mice. Cells from the first and second lavages 1‐2, and from lavages 3‐4, 5‐6, 7‐8, and 9‐10 were stored in saline‐EDTA at 4°C, and total cells were counted in a hemacytometer (Neubauer). Data show that lavage must be done 10 times for the most efficient cell recovery. Dots are means and bars are SEM values ( n = 6) View Image -
Figure 2. Evolution over time in the total number of cells counted in BALF from control (Ctrl 1), OVA‐sensitized and ‐challenged (OVA 1), or LPS‐treated (LPS 1) mice. Cells were stored in saline‐EDTA at 4°C; total cells were counted in a Neubauer hemacytometer on days 0, 1, 2, and 3. Data show that total cell count should be performed within 24 hr after BAL. Dots are means and bars are SEM values ( n = 3.) View Image -
Figure 3. Evolution over time of differential cell counts on cytospinning after BAL storage at 4°C. Cells were stored in saline‐EDTA at 4°C and cytospun and stained on days 0, 1, 2, 3, 4, or 7. The data show that cells (A ) can be stored a few days to avoid immediate cytospinning but that they must be cytospun within 3 days after the BAL for correct differential cell count. After more than 3 days, the cells become fragile, and fewer eosinophils (C ), neutrophils (D ) and lymphocytes (E ) are preserved on cytospinning, resulting in an increased percentage of the more “stable” macrophages (B ) after 3 days. Dots are means and bars are SEM values ( n = 3). View Image -
Figure 4. Photograph of cytospun BAL cells stained with Diff‐Quick. (A ) Control BAL; (B ) BAL from ovalbumin‐sensitized and challenged mice. BAL cells show macrophages, eosinophils, neutrophils and lymphocytes in OVA‐challenged animals. View Image -
Figure 5. Interindividual reproducibility of total cell counts. Cells in saline‐EDTA were cytospun and counted on a hemacytometer after BAL. Dots are individual values and horizontal bars are means ( n = 6). View Image -
Figure 6. Interindividual reproducibility of differential cell counts. Cells in saline‐EDTA were cytospun and stained before the differential cell count; 100 (right panel) or 400 (left panel) cells were counted and identified on six replicated cytospins. Data show that counting and identifying 400 cells is necessary and sufficient for good reproducibility. Dots are individual values and horizontal bars are means ( n = 6). View Image
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. | |
| Blé, F.‐X, Cannet, C., Zurbruegg, S., Gérard, C., Frossard, N., Beckmann, N., and Trifilieff, A. 2009. Activation of the lung S1P1 receptor reduces allergen‐induced plasma leakage in mouse. Brit. J. Pharmacol. 158:1295‐1301. | |
| Delayre‐Orthez, C., Becker, J., De Blay, F., Frossard, N., and Pons, F. 2004. Dose‐dependent effects of endotoxins on allergen sensitization and challenge in the mouse. Clin. Exp. Allergy 34:1789‐1795. | |
| Delayre‐Orthez, C., Becker, J., Guenon, I., Lagente, V., Auwerx, J., Frossard, N., and Pons, F. 2005a. PPARalpha downregulates airway inflammation induced by lipopolysaccharide in the mouse. Respir. Res. 6:91. | |
| Delayre‐Orthez, C., Becker, J., de Blay, F., Frossard, N., and Pons, F. 2005b. Exposure to endotoxins during sensitization prevents further endotoxin‐induced exacerbation of airway inflammation in a mouse model of allergic asthma. Int. Arch. Allergy Clin. Immunol. 138:298‐304. | |
| Delayre‐Orthez, C., Becker, J., Auwerx, J., Frossard, N., and Pons, F. 2008. Suppression of allergen‐induced airway inflammation and immune response by the peroxisome proliferator‐activated receptor‐alpha agonist fenofibrate. Eur. J. Pharmacol. 581:177‐184. | |
| Deschamps, K., Cromlish, W., Weicker, S., Lamontagne, S., Huszar, S.L., Gauthier, J.Y., Mudgett, J.S., Guimond, A., Romand, R., Frossard, N., Percival, M.D., Slipetz, D., and Tan, C.M. 2011. Genetic and pharmacological evaluation of Cathepsin S in a mouse model of asthma. Am. J. Respir. Cell Mol. Biol. 45:81‐87. | |
| Hachet‐Haas, M., Balabanian, K., Rohmer, F., Pons, F., Franchet, C., Lecat, S., Chow, K.Y., Dagher, R., Gizzi, P., Didier, B., Lagane, B., Kellenberger, E., Bonnet, D., Baleux, F., Haiech, J., Parmentier, M., Frossard, N., Arenzana‐Seisdedos, F., Hibert, M., and Galzi, J.‐L. 2008. Small neutralizing molecules to inhibit actions of the chemokine CXCL12. J. Biol. Chem. 283:23189‐23199. | |
| Henderson, A.J.W. 1994. Bronchoalveolar lavage. Arch. Dis. Childhood 70:167‐169. | |
| Hunninghake, G.W., Gadek, E., Kawanami, O., Ferrans, V.J., and Crystal, R.G. 1979. Inflammatory and immune processes in the human lung in health and disease: Evaluation by bronchoalveolar lavage. Am. J. Pathol. 97:149‐206. | |
| Mathers, R.A., Evans, G.O., Bleby, J., and Tornow, T. 2006. Total and differential leucocyte counts in rat and mouse bronchoalveolar lavage fluids using the Sysmex XT‐2000iV. Comp. Clin. Path. 16:29‐39. | |
| 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. | |
| Van Rijt, L.S., Kuipers, H., Vos, N., Hijdra, D., Hoogsteden, H.C., and Lambrecht, B.N. 2004. A rapid flow cytometric method for determining the cellular composition of bronchoalveolar lavage fluid cells in mouse models of asthma. J. Immunol. Methods 288:111‐121. | |
| Zhang, Z., Hener, P., Frossard, N., Kato, S., Metzger, D., Li, M., and Chambon, P. 2009. Thymic stromal lymphopoietin overproduced by keratinocytes in mouse skin aggravates experimental asthma. Proc. Natl. Acad. Sci. U.S.A. 106:1536‐1541. |
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library
