Evaluation of Inflammatory Response in Murine Lung

Anesthetized mice were euthanized by exsanguination at days 1, 3 and 7 after CNP instillation. Blood was withdrawn from the retro orbital plexus with a capillary and collected in EDTA coated tubes (Sarstedt, Germany) for hematological analysis (ADVIA Hematology Systems (Bayer Diagnostics). Lungs were lavaged with 10 × 1 mL phosphate buffered saline (PBS, calcium and magnesium free) as described previously [38 (link)]. The BAL fluids from the first two lavages were used for protein analysis after cell separation by centrifugation (425 × g, 20 min at room temperature). The cell pellet was resuspended in 1 mL RPMI 1640 medium (BioChrome, Berlin, Germany) supplemented with 10% fetal calf serum (Seromed, Berlin, Germany). Cell viability was determined by the Trypan blue dye exclusion test. Differential cell counts were performed on cytospin preparations (May-Grünwald-Giemsa staining; 2 × 200 cells were counted).