3 3 5 5 tetramethylbenzidine tmb substrate reagent set

To obtain extracellular proteins from USA300 strains, supernatant from S. aureus sub-cultured for 5 h was sterile-filtered (0.22 um) and stored at -80°C. For human myeloperoxidase (MPO) activity assays, 0.5 ug of recombinant human MPO (R&D Systems) in 50 uL of DPBS was mixed with 50 uL of sterile-filtered S. aureus supernatant for 30 min at room temperature. The MPO-supernatant solution was then exposed to 150 uL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate reagent set (BD Biosciences) and incubated at 37°C. The oxidation of TMB catalyzed by human MPO was quantified by measuring the OD₆₅₀ every minute for 30 min using a SpectraMax Paradigm microplate reader (Molecular Devices). The MPO inhibitor sodium azide was used at a concentration of 1 mM.

Concentrations of IL-6 in the cell culture supernatants were measured by commercially available human IL-6 ELISA set (BD Biosciences, Palo Alto, CA). The ELISAs were performed according to manufacturer’s instructions. Flat-bottom 96-well microplates were coated with anti-human IL-6 monoclonal antibody at 4 °C overnight. Cell supernatants were assayed as neat or one- to fourfold dilutions. 3,3′,5,5′-tetramethylbenzidine (TMB) substrate reagent set (BD Biosciences) was used to detect the streptavidin–horseradish peroxidase (BD Biosciences) reaction and optical densities were measured at 450 nm with 750 nm wavelength corrections in a microplate reader. The experiments were carried out in triplicate wells in at least three independent experiments.