4.5. Differential Cell Count in Bronchoalveolar Lavage (BAL) Fluid

Before explantation of airways or trachea, the trachea was cannulated and the lungs were rinsed with 1 mL D-PBS (Sigma-Aldrich Chemie GmbH, Munich, Germany) containing a protease inhibitor (Complete, Roche, Basel, Switzerland). The volume of BAL fluid was recorded and the number of immune cells was determined using a Neubauer counting chamber. For differential cell count, 50 µL of BAL fluid was cytospinned (500× g, 5 min; Cytospin™, Thermo Scientific GmbH, Schwerte, Germany) and the immune cells (macrophages, lymphocytes, eosinophil and neutrophil granulocytes) were stained using the Pappenheim method or the Diffquick solutions (Medion Grifols Diagnostics AG, Düdingen, Switzerland). A total number of at least 100 immune cells was counted. The results are presented as the total number of macrophages, eosinophils, neutrophils and lymphocytes per mL BAL fluid.