O dianisidine reagent

To quantify macrophage efferocytosis of apoptotic neutrophils, polymorphonuclear cells (1 × 10⁴ cells/mL) were incubated with either control medium (10% HI-FBS in HBSS), tylvalosin (0.1, 1.0, or 10 µg/mL), lincomycin (11.3 µM), or the positive control for apoptosis staurosporine (1 µM), for 30 min (23 (link)). Following treatment, cells were washed with HBSS (free of serum), centrifuged at 850 g for 5 min and resuspended in warm IMDM containing 10% HI-FBS and cocultured with monocyte-derived macrophages for 2 h at 37°C in a humidified incubator. Both the supernatant fraction containing free neutrophils, and the coculture monolayer fraction, were lysed with lysis buffer (1:1 ratio of 1 M citrate and 10% Triton-X 100; Sigma-Aldrich) and incubated for 15 min at 4°C while shaking. Myeloperoxidase activity, as a marker of neutrophil accumulation, was measured in coculture supernantants and monolayers with a kinetic assay, as described previously (12 (link)). Immediately before reading the kinetic absorbance, o-dianisidine reagent (Sigma) was added. Absorbance readings were taken at 460 nm once every 30 s for 16 min using a SpectraMax M2e microplate reader (Molecular Devices). Enzyme activity was defined as the change in optical density over time (mU/min).

Catalase (CAT) activity was performed after diluting the sample (1:100) in 50 mM phosphate buffer. The method involves two reactions: (1st) H₂O₂ (10 nM) undergoes dismutation by tissue catalase for 10 min at room temperature. This reaction is stopped by the addition of NaN₃ (1 mM); (2nd) the remaining H₂O₂ is determined by oxidation of the o-dianisidine reagent (OD; 0.167 mg/mL; Sigma; St. Louis, MO, USA) in a reaction catalyzed by the enzyme peroxidase HRP (horse radish peroxidase; 0.095 mg/mL; Sigma; St. Louis, MO, USA), at pH 6.0. The speed of the o-dianisidine oxidation product was monitored by the increase in absorbance at 460 nm (Spectra max Plus 384, Molecular Devices Inc.; Sunnyvale, CA, USA) for 10 min. In order to inactivate the catalase (reaction blank), supernatants were incubated at 60 °C for 2 h. The catalase activity value was calculated from the maximum speed per minute of each reaction and extrapolated on the H₂O₂ curve. The standard H₂O₂ curve (8820–11.3 μM) was performed and the results were expressed in catalase units (UCAT)/mg protein. A catalase unit was defined as the degradation of 1 µmol of H₂O₂ min⁻¹ at 25 °C.