Bronchoalveolar lavage procedure

The study of BAL cell composition was used to assess lung inflammation. BAL cells were recovered by flushing the lungs four times with 0.8 mL of physiological saline using a tracheal cannula. The BAL fluids recovered were pooled and centrifuged (500 g, 10 min, 4 °C). The supernatant was stored for later analyses and the pellet was re-suspended in a 100 μL PBS buffer containing heparin (20 IE/mL) and serum albumin (0.003%). The total numbers of cells were determined using a hemocytometer. For differential counts, cytospin preparations were made (Cytospin®2, StatSpin® Inc., Norwood, MA) (1000 g, 4 min, RT). Slides were stained with May-Grünwald/Giemsa and all slides were inspected in a blinded manner by the same technician. Cells were identified by standard morphology and differentiated into neutrophils, eosinophils, epithelial cells, lymphocytes, and macrophages. For each slide, 200 cells were counted.