Antibiotic and Phenazine Compound Preparation

Non-pharmaceutical-grade antibiotics included colistin sulfate (∼20,000 U/mg; Chem-Impex), colistin sodium methanesulfonate (CMS) (from Bacillus colistinus; Fluka), and polymyxin B sulfate (∼8,000 U/mg; Research Products International). Stock solutions of CMS were kept frozen until use. The reagent-grade phenazines 1-hydroxyphenazine (1-HP), phenazine-1-carboxylic acid (PCA), and phenazine-1-carboxamide (PCN) were obtained from ArkPharm and were used without further purification. Crystalline pyocyanin was purchased from Cayman or prepared by photolysis of a 2 mM solution of phenazine methosulfate in 20 mM HEPES buffer (pH 7.4), as reported previously (32 (link)). In-house nanopure water was generated by double distillation and used in all experiments. Antioxidants employed in this study included N-acetyl-l-cysteine (NAC), tyrphostin AG490, chlorogenic acid (ChlA), deferasirox (Dfx), the cell-permeable catalase/SOD mimic EUK134, o-phenanthroline (o-Phen), and resveratrol (Resv).