Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

The overall goal of this procedure is to harvest the cellular and acellular contents of the lung lumen via broncoalveolar lavage for ex vivo analyses and for assessment of the ongoing disease state of the animal. This method can help answer key questions in the respiratory immunology field about infection and inflammation induced immune mechanisms through the quantification of innate cellular and humoral responses in the lung. The main advantages of this technique are that it is simple, efficient, and highly reproducible and there is no need of special equipment or tools.

Before beginning the procedure, create a catheter by inserting a 23 gauge needle into a 0.5 centimeter piece of transparent plastic polyethylene 21 gauge tubing and place the animal in the supine position. Secure the mouse's limbs and disinfect the neck with 70%ethanol. Then use a scalpel to make a skin incision near the trachea.

Open the skin to expose the salivary glands and use forceps to separate the glands and to reveal the sternal hyoid muscle. Use the forceps to incise the muscle around the trachea. When it is visible, use the forceps to thread a cotton suture under the tracheal tissue.

Next, use a 26 gauge needle to carefully puncture the middle of the trachea between two cartilage rings and insert the pre-made catheter about 0.5 centimeters into the tracheal lumen. Stabilize the catheter with the suture. Then, load a one milliliter syringe with one milliliter of sterile balanced salt solution supplemented with 100 micromolar EDTA.

Connect the loaded syringe to the catheter and gently inject the balanced salt and EDTA solution into the lung. When all of the saline has been injected, gently aspirate the solution while massaging the thorax. It's very important to be gentle when injecting and retaking the in solid fluid to maximize BAL fluid retrieval and to minimize sheering forces.

If the the in solid fluid goes through the nose, re-secure the suture along the catheter. When all of the aspirate has been collected, transfer the fluid into a 15 milliliter tube on ice and repeat the lavage two more times. Once mastered, this technique can be completed in seven minutes if it is performed properly.

After the third bronchoalveolar lavage collection, centrifuge the pooled aspirate and store the supernatant at negative 80 degrees celsius. Re-suspend the pellet in 200 microliters of ACK lysis buffer. After no more than two minutes at room temperature, dilute the lysis buffer with one milliliter of cold PBS and collect the cells by centrifugation.

Re-suspend the pellet in the appropriate volume of PBS for the number of samples to be analyzed and aliquot the cells into the appropriate number of wells of a 96 well plate for the analysis. Collect the cells by another centrifugation and re-suspend the pellets in 50 microliters of FC Block and PBS. After 10 minutes at room temperature, add 50 microliters of the appropriate antibody cocktail of interest to each well and incubate the cells for 30 minutes at 4 degrees celsius protected from light.

At the end of the incubation, centrifuge the plate and discard the supernatant, re-suspending the pellets to a final volume of 200 microliters of FAX buffer per well for flow cytometric analysis. Using a gating strategy based on the differential expression of antigens on the surfaces of various bronchoalveolar lavage cell populations, it is possible to identify a variety of immune cell types. The cells and singlets are gated according to their forward and side scatter properties.

The live/dead dye-negative cells are gated, followed by the identification of the Cd11c High and Cd11c Low cells. In the Cd11c High population, macrophages and dendritic cells can be identified based on their MHC-II and SinglecF expression respectively. In the Cd11c Low population, T cells and B cells can be identified based on their respective CD3/CD19 and MHC-II expression.

In the remaining cells, eosinophils and neutrophils can be identified by their Cd11b or Ly-6G marker expression respectively. Counting beads can also be added to determine the absolute cell numbers of the different cell populations by comparing the ratio of bead to cell events, for example, in naive versus LPS stimulated animals. After harvesting the BAL fluid, other techniques like ELISA immunoblot cytokine bead array can be performed to answer additional questions about the acellular content of the BAL fluid.

After its development, this technique paved the way for researchers of the immunology field to explore the cellular and acellular content of the lung lumen of mice. After watching this video, you should have a good understanding how to efficiently and reproducibly perform BAL.