Murine Bronchoalveolar Lavage

Materials and Reagents

1. Needles (BD, catalog number: 305167 ) or tapes
   
2. Nylon string (Dynarex, catalog number: 3243 )
   
3. 22 G x 1” Exel Safelet Catheter (Exel International, catalog number: 26746 )
   
4. 1 ml syringe (BD, catalog number: 309659 )
   
5. 1.5 ml Eppendorf tubes (VWR, catalog number: 87003-294 )
   
6. 0.22 µm filter (EMD Millipore, catalog number: SLGP033RS )
   
7. Mice (THE JACKSON LABORATORY)
   
8. 70% ethanol (Decon Labs, catalog number: 2701 )
   
9. Protease inhibitor cocktail (Roche Diagnostics, catalog number: 11697498001 )
   
10. Phenylmethylsulfonyl fluoride (PMSF) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 36978 )
    
11. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9625 )
    
12. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
    
13. Disodium hydrogen phosphate heptahydrate (Na₂HPO₄·7H₂O) (Fisher Scientific, catalog number: BP331-500 )
    
14. Potassium phosphate monobasic (KH₂PO₄) (Acros Organics, catalog number: 205925000 )
    
15. Ammonium chloride (NH₄Cl) (Sigma-Aldrich, catalog number: A9434-1KG )
    
16. Potassium bicarbonate (KHCO₃) (Sigma-Aldrich, catalog number: P9144-1KG )
    Note: This product has been discontinued.
    
17. Ethylenediaminetetraacetic acid disodium salt dihydrate (Na₂-EDTA-2H₂O) (Sigma-Aldrich, catalog number: E5134-100G )
    
18. Phosphate-buffered saline (PBS) (see Recipes)
    
19. ACK lysis buffer (see Recipes)
    

Equipment

1. CO₂ chamber
   
2. Biosafety cabinet
   
3. Styrofoam board
   
4. Forceps (Roboz Surgical Instrument, catalog number: RS-5135 )
   
5. Scissors (Roboz Surgical Instrument, catalog number: RS-6802 )
   
6. Centrifuge (Eppendorf, model: 5417 R )
   
7. Hemocytometer (Hausser Scientific, catalog number: 3110 )
   
8. Microscope (Olympus, model: CK30 )
   

Procedure

1. Sacrifice a mouse by CO₂ inhalation in a CO₂ chamber.
   
2. Bring the mouse to a biosafety cabinet.
   
3. Dampen the mouse with 70% ethanol.
   
4. Place the mouse front side up on styrofoam panel and fix the arms and legs with needles or tapes (Figure 1A).
   
5. Use scissors to make incision in the skin from abdomen to neck, and tear skin with forceps to expose thoracic cage and neck (Figure 1A).
   
6. Gently remove the muscle around the neck to expose trachea (Figure 1B).
   
7. Use forceps to put a ~10 cm-long nylon string under the trachea (Figure 1C).
   
8. Carefully cut the ribs to expose the heart and the lungs (Figure 1D).
   Note: Do not cut the trachea and lungs.
   
9. Insert a 22 G x 1” Exel Safelet Catheter into the trachea, remove the stylet hub, and tie the catheter and the trachea together firmly with a nylon string (Figures 1E-1G).
   
10. Load a 1 ml syringe with 0.8 ml of PBS and place it in the end of the catheter (Figure 1H).
    
    
    Figure 1. Mouse bronchoalveolar lavage steps
    
    
11. Slowly inject and aspirate the PBS 4 times, remove the syringe from catheter, and save the recovered lavage fluid in 1.5 ml Eppendorf (EP) tubes on ice.
    
12. Repeat steps 10-11 for additional three times.
    
13. Record BAL volume according to the scales on the 1.5 ml EP tubes.
    
14. Centrifuge at 800 x g for 10 min at 4 °C.
    
15. Transfer the supernatant to a new 5 ml tube, add protease inhibitor cocktail to a final concentration of 1x and PMSF to a final concentration of 1 mM, and mix well. Store at -80 °C if not used immediately. Pathogens, proteins such as cytokines/chemokines, and other components in the supernatants of BAL fluid (BALF) can be analyzed by bicinchoninic acid protein assay, ELISA, luminex assays, and/or other assays.
    
16. Resuspend BAL cell pellets in 200 µl of PBS and combine the cell suspensions.
    
17. Centrifuge at 800 x g for 10 min at 4 °C.
    
18. Resuspend BAL cell pellets in 100-200 µl ACK lysis buffer, lyse red blood cells on ice for 10 min, and then add 1 ml of PBS to stop lysis.
    
19. Centrifuge at 800 x g for 10 min at 4 °C.
    
20. Resuspend BAL cell pellets in 400 µl of PBS, count the cells by taking about 20 µl of the cell sample to a hemocytometer and counting the cells under a microscope. Record the cell number. These BAL cells can then be used for FACS analysis, spun onto slides for staining (Figure 2), and/or lysed for RNA or protein extraction.
    
    
    Figure 2. Hema3-staining of immune cells in murine BAL. White arrow: macrophage; grey arrow: lymphocyte; black arrow: neutrophil. Scale bar = 10 µm.
    

Data analysis

Murine BAL cells are retrieved and cytospun onto slides, which are Hema3-stained to determine the immune cells.

Notes

Although pelleted BAL cells from all four washes are combined to analyze the cells, you can combine the supernatant from only the first two washes (Procedure step 11) to analyze the BAL fluid components, as the first two washes contain a high concentration of these components.

Recipes

1. Phosphate-buffered saline (PBS) (1 L)
   8.0 g NaCl
   0.2 g KCl
   1.15 g Na₂HPO₄·7H₂O
   0.2 g KH₂PO₄
   Adjust to pH 7.4
   
2. ACK lysis buffer (1 L)
   1 L deionized water
   8.29 g NH₄Cl
   1 g KHCO₃
   37.2 mg Na₂-EDTA
   Adjust pH to 7.2-7.4, sterilize using 0.22 µm filter, store at 4 °C
   

Acknowledgments

The authors thank the members in Xiao-Qu lab for the helpful discussion. This study was supported in part by the National Institute of Health (NIH)/National Cancer Institute (NCI) grants R01 CA172090, R21 CA175252, P50 CA090440, P30 CA047904, as well as the American Lung Association (ALA) Lung Cancer Discovery Award LCD 259111 and American Cancer Society (ACS) Fellowship Award PF-12081-01-TBG.

References

1. Daubeuf, F. and Frossard, N. (2012). Performing bronchoalveolar lavage in the mouse. Curr Protoc Mouse Biol 2(2):167-175.
   
2. Qu, Z., Sun, F., Zhou, J., Li, L., Shapiro, S. D. and Xiao, G. (2015). Interleukin-6 prevents the initiation but enhances the progression of lung cancer. Cancer Res 75(16): 3209-3215.
   
3. Sun, F., Qu, Z., Xiao, Y., Zhou, J., Burns, T. F., Stabile, L. P., Siegfried, J. M. and Xiao, G. (2016). NF-κB1 p105 suppresses lung tumorigenesis through the Tpl2 kinase but independently of its NF-κB function. Oncogene 35(18): 2299-2310.
   
4. Zhou, J., Qu, Z., Sun, F., Han, L., Li, L., Yan, S., Stabile, L. P., Chen, L. F., Siegfried, J. M. and Xiao, G. (2017). Myeloid STAT3 promotes lung tumorigenesis by transforming tumor immunosurveillance into tumor-promoting inflammation. Cancer Immunol Res 5(3): 257-268.
   
5. Zhou, J., Qu, Z., Yan, S., Sun, F., Whitsett, J. A., Shapiro, S. D. and Xiao, G. (2015). Differential roles of STAT3 in the initiation and growth of lung cancer. Oncogene 34(29): 3804-3814.