Elastin congo red dye

The used P. aeruginosa PAO1 strain was donated by the Department of Microbiology, Faculty of Pharmacy, Mansoura University. Microbiological media, Mueller Hinton (MH) broth, Tryptone soya broth (TSB) and agar, and Luria-Bertani (LB) broth and agar were purchased from Oxoid (Hampshire, UK). All chemicals were of pharmaceutical grade. Sotolon, and elastin Congo red dye were obtained from Sigma (St. Louis, MO, USA).
To show the anti-virulence activities of Sotolon, a single pure colony of PAO1 was selected and grown OVN with shaking to O.D600 0.4, which equivalates to approximately 1 × 10⁶ CFU/mL. Then, the bacterial suspensions were added to suitable media (according to each experiment) with Sotolon in sub-MIC, or control without Sotolon. Our results were normalized in all of the experiments by using a fixed bacterial inoculum size (1 × 10⁶ CFU/mL).
According to a Sotolon supplier, it was provided as 10 wt.% in propylene glycol. To exclude any effect of propylene glycol, we treated the PAO1 strain with propylene glycol in equivalent concentrations to those used with Sotolon (1/4 or 1/8 MIC). It is worthy to mention that propylene glycol in the used concentrations did not have any significant influence on bacterial growth or bacterial virulence (data not shown).

MPO and ELA activities were measured in the supernatants from lesions. Lesion sample supernatants were obtained by disaggregating the tissue through treatment with RPMI 1640 medium containing 0.25% collagenase (Worthington) and were frozen at -80°C until analysis. The total protein concentration was measured using a Quick Start Bradford Protein assay kit (Bio-Rad, CA, USA). MPO activity in the supernatants was assayed by measuring the change in absorbance at 450 nm using 3,3′,5,5′ tetramethylbenzidine (50%) and H₂O₂ (50%) (Opteia Set B; BD Biosciences, San Diego, CA, USA). The reaction was stopped after 15 min using phosphoric acid (1M).
For the ELA analysis, supernatants were individually incubated with insoluble elastin-Congo Red dye (E0502-5G; Sigma- Aldrich) diluted in 100 mM Tris–HCl buffer (pH 8.8) containing 0.01% sodium azide (Sigma- Aldrich) in a 96-well plate. The optical absorption of the hydrolysis product was read with a Microplate Reader (Bio-Rad Model 680; Bio-Rad Laboratories, Hercules, CA, USA) at 450 nm after 24 h incubation at 37°C. ELA activity was calculated from a standard curve of elastase from human leucocytes (Sigma-Aldrich) and expressed in units/ml.