Bronchoalveolar lavage fluid (BALF) collection and analysis

At 6 h after intraperitoneal injection of LPS, rats were anesthetized before a plastic cannula was inserted into the trachea to collect BALF by sterile physiological saline aspiration, which was performed three times. The rats were then euthanized by exsanguination until cardiac arrest. The BALF sample was centrifuged at 1,300 x g at 4˚C for 10 min and the supernatant was extracted. Bicinchoninic acid protein assay (BCA) method was used to detect total protein concentration in the BALF samples, whilst the cell pellet was resuspended in PBS. The cells were then stained with Wright-Giemsa stain for 1 min at room temperature and the total cell counts were assessed using a cell count chamber and observed under a light microscope under a x4 objective lens.