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.