Multiwell plate reader

To analyse growth curve, P. aeruginosa PA14 wild-type cultures were grown in the presence of increasing concentrations of prodigiosin (20–500 μM) and copper (20–200 μM). Optical density was measured at 600 nm using multiwell plate reader (Perkin Elmer, USA).
P. aeruginosa PA14 biofilm was grown in 300 μl of LB₁₀ in a 96-well plate supplemented with/without prodigiosin (500 μM) and Prd/Cu(II) (100 μM). Cultures were incubated overnight at 37°C in a shaking incubator at 120 rpm. Propidium iodide (PI) was used to stain nucleic acids in biofilms. PI was dissolved in distilled water at a final concentration of 6 μM. Each well was incubated with 200 μl of PI solution over 30 min at room temperature and fluorescence (535/620 nm) was measured using multiwell plate reader (Perkin Elmer, USA). Washed biofilms were treated with DNaseI and RNaseA (Qiagen, USA) over 30 min at 37°C, where it is required. All cultures were prepared in triplicates.

Hydrogen peroxide (H₂O₂) production from prodigiosin and copper (II) acetate (Sigma, USA) treated samples were analyzed by Amplex Red Hydrogen Peroxide assay kit (ThermoFisher Scientific, USA). Assay buffer (pH: 7.4) provided by the kit was supplemented with different concentrations of prodigiosin and copper. EDTA (10 mM) was used as metal chelator. H₂O₂ release was monitored over 6 h at 37°C according to manufacturer‘s instructions. Absorbance readings were taken by a multiwell plate reader (Perkin Elmer, USA). All samples were prepared in triplicates.