Bronchoalveolar lavage and lung collection
Animals were euthanised at the end of the experimental protocol via
intraperitoneal injection of sodium pentobarbitone (240 mg
kg-1; Virbac, NSW, Australia). Lungs were then lavagedin situ via a surgical tracheotomy with 0.4 mL of chilled PBS
initially followed by 0.3 mL PBS thrice, with ~1mL of
BAL fluid (BALF) retrieved per mouse as previously described (Vlahos et
al., 2006) . Total viable cell numbers in the BALF were determined using
50μL of BALF diluted with 50 µl of acridine orange/ethidium bromide
(AO/EB) (Invitrogen, USA). Cell counting was carried out using a
standard Neubauer haemocytometer, under fluorescent light on an Olympus
BX53 microscope (Olympus, Japan). Right ventricular perfusion with 6-7
mL of PBS was then performed to clear whole lungs from blood, and the
lungs then excised, rinsed in PBS, snap-frozen in liquid nitrogen and
stored at -80°C until required.