Varioscan lux multimode plate reader

The mitoSOX red fluorescent probe was utilized for the assessment of mitochondrial derived oxygen reactive species [34 ]. The assay was performed seeding 5000 MCF-7 cells on a 96-wells culture plate. After 24 h, the cells were exposed to 1 µM doxorubicin (positive control), 28 μM EP or 1% DMSO containing cell culture media for 48 h. Then, the cells were washed twice with PBS and exposed to mytoSOX for 10 min; next, they were washed twice again with PBS and the fluorescence detected at 510/595 nm of excitation and emission, respectively, with a Varioscan™LUX multimode plate reader (Thermoscientific). Next, the protein content was determined by using the sulforhodamine B (SRB) assay. For this, the cells were fixed using cold 1% acetic acid in methanol and then exposed to SRB 0.5% w/v for 1 h at 37 °C. After removing the SRB, the wells were gently washed with 1% acetic acid, then the plate was dried and the fixed dye solubilized by adding 200 μL of 10 mM tris pH 10. The absorbance was read with the Varioscan™LUX multimode plate reader (Thermoscientific) at 580 nm. The results were expressed as relative fluorescence units normalized by the protein content of each well.

The E. coli and P. aeruginosa strains used in this study are listed in Table S1 in the Supplementary Materials and the plasmids in Table 3. Bacteria were grown routinely in Luria-Bertani (L-broth) medium or on L-agar (L-broth with 1.5% (wt/vol) agar) at 37 °C. To determine growth on different sulphur sources, P. aeruginosa strains were grown in modified M9 minimal medium (33.7 mM Na₂HPO₄, 22 mM KH₂PO₄, 8.55 mM NaCl, 9.35 mM NH₄Cl, 1.0 mM MgCl₂, 0.3 mM CaCl₂, 152 mM leucine, 134 µM FeCl₃, 20 mM sodium citrate), and supplemented with appropriate sulphur sources (0.5 mM). Growth curves were obtained with the use of a Varioscan Lux multimode plate reader (Thermo Scientific™) in 96-well plates.
Where needed, appropriate antibiotics were added to the media as follows: ampicillin, 100 µg mL⁻¹ for Ap^R in E. coli, kanamycin sulphate, 50 µg mL⁻¹ for Km^R in E. coli, 25 µg mL⁻¹ chloramphenicol for Cm^R in E. coli, and 200 µg mL⁻¹ in P. aeruginosa; carbenicillin disodium salt, 300 µg mL⁻¹ for Cb^R in P. aeruginosa; rifampicin, 300 µg mL⁻¹ for Rif^R in P. aeruginosa.