Quantification of Lung Neutrophil Infiltration

Assessment of inflammation and infiltration of neutrophils during mono or coinfections of mouse lung tissues were evaluated by quantification of MPO activity using the o-dianisidine-H₂O₂ method, as previously described (Côté-Gravel et al., 2016 (link)). Briefly, 10 μl of lung homogenate was mixed with a solution of o-dianisidine hydrochloride (167 μg/ml) (Sigma–Aldrich, Oakville, ON, Canada), 0.0005% H₂O₂ (Sigma–Aldrich, Oakville, ON, Canada), 50 mM hexade-cyltrimethylammonium bromide (CTAB) and 50 mM phosphate buffer at pH 6.0, in a 96-well plate. The A_460nm was then measured at intervals of 15 s for 8 min and the maximum reaction rate was considered. One unit of MPO was defined as the quantity of enzyme degrading 1 μmol of H₂O₂/min at 25°C, with an absorption coefficient of 11.3 mM^–1 cm^–1 at 460 nm for o-dianisidine. MPO units were normalized according to the lung weight.