Tissue collection
The lungs were lavaged in situ using 0.4ml of ice-cold PBS and three subsequent repeats of 0.3ml PBS, with a return of approximately 1ml of bronchoalveolar lavage fluid (BALF) per mouse as previously published (Chen et al., 2006; Vlahos et al., 2006). 20μL of BALF was diluted 1:1 with Acridine Orange and the total number of viable cells counted on a standard Neubauer haemocytometer under fluorescent light on an Olympus BX53 microscope (Olympus, Japan). To differentiate cell populations in BALF, cytocentrifuge preparations (Shandon Cytospin 3, 400 rpm, 10 min) were performed using approximately 5x104 cells from BALF. Once dried, cells were fixed with Shandon™ Kwik-Diff™ fixative (Thermo Fischer Scientific, USA) and subsequently stained with Hemacolor® Rapid Red and Blue dye (Merck, Germany) as per manufacturers’ instructions, mounted with Enetellan® new (Merck, Australia). Cell types (macrophages, lymphocytes, and neutrophils) were identified according to standard morphological criteria and at least 500 cells per slide were counted. After the lavage procedure, 10ml of PBS was used to clear the lungs of blood via a right ventricular perfusion of the heart. Lungs were then weighed, snap frozen in liquid nitrogen and stored at -80°C until required. Lower limb muscles were removed tendon to tendon from each mouse. The muscles were weighed, snap frozen in liquid nitrogen and stored at -80°C until required.