O dianisidine hydrochloride

MPO activity was assayed as an index of neutrophil infiltration in mouse lung that was subjected to LPS-induced ALI. Frozen lung tissues were thawed and immersed in 50 mM phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide. Lung tissue samples were sonicated on ice using a homogenizer, and the homogenate was then centrifuged at 2,000 g for 15 min at 4°C. The MPO activity of homogenates was determined by adding them to phosphate buffer (pH 6.0) containing 0.167 mg/mL o-dianisidine hydrochloride and 0.0005% hydrogen peroxide (Sigma). The light absorbance was monitored at 460 nm by a spectrometer for 5 min according to the kit instructions. MPO activity was calculated by comparing results with the standardized concentration curve derived from commercial human MPO (Sigma). Final values were all normalized to the corresponding protein concentration. A Bio-Rad assay kit (Hercules, CA) was used to detect the protein content of every sample. Lung MPO activity was finally expressed in units per gram of tissue.

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.

The samples of midbrain tissue were rapidly removed and frozen in liquid nitrogen. We measured out an appropriate amount of frozen tissue, washed it twice in phosphate buffer, pH 6.0 at 4–8 °C, and then homogenised it (IKA Homogenizer, Staufen, Germany) in a solution of 0.5% hexadecyltrimethylammonium bromide (HTAB) mixed in 50 mM potassium phosphate buffer (pH 6). The sample underwent centrifugation for 20 min at a temperature of 4 °C and 10,000 revolutions per minute. The samples were sonicated for 20 s, then frozen and thawed thrice. A 2.9 mL solution of 50 mM potassium phosphate buffer at pH 6 containing 0.167 mg/mL of O-dianisidine hydrochloride and 0.0005% H₂O₂ was allowed to react with a 0.1 mL aliquot of the supernatant or standard (Sigma, Germany). After 5 min, the reaction was stopped with 0.1 cc of 1.2 M hydrochloric acid. The absorbance rate at 460 nm was measured using a spectrophotometer (Cecil 9000, Cambridge, UK). MPO activity was expressed as milli-units (mU) per gram weight of moist tissue [71 (link)].