Infinite 200 pro multi well plate reader

The PEP Colorimetric/Fluorometric Assay Kit from Sigma-Aldrich was used to determine the PEP concentration in S. coelicolor according to the manufacturer’s protocol. 2–4 mg of mycelium harvested from solid medium were used for the enzymatic reaction. Signals were quantified by fluorescence using a Tecan infinite 200 pro multiwell plate reader.

To examine cell proliferation ability, cells were seeded on silicone plates at a density of 110 cells/mm² and cultured in the growth medium as previously indicated. The cell number at each time-point was counted with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) colorimetric assay (Merk Life Science s.r.l., Milano, Italy). Briefly, 20 μL of MTT solution (5 mg/mL in PBS w/w Ca²⁺ and Mg²⁺) was added to each well followed by 2 h of incubation at 37 °C with 5% CO₂. Then, 100 μL of extraction buffer (5% SDS in N,N-Dimethylformamide) was added to each well followed by 2 h of incubation at 37 °C with 5% CO₂. In total, 150 μL per silicone well was transferred to a 96-well plate to measure the optical density of the formazan products. The absorbance of the formazan products was recorded at 570 nm using an Infinite^® 200 PRO multi-well plate reader (Tecan Group Ltd., Männedorf, Switzerland) (Supplementary Figure S7).

Stationary-phase bacteria were diluted 1000-fold in fresh CA-MHB, and 4 μl was seeded into the wells of a microtitre plate containing 200 μl CA-MHB, and for some experiments the LptD inhibitor murepavadin, to give a final inoculum of 5 × 10⁵ c.f.u. ml⁻¹. The microtitre plate was incubated with shaking (180 r.p.m.) at 37°C for 16 hr in a Tecan Infinite 200 Pro multiwell plate reader (Tecan Group Ltd., Switzerland) and optical density measurements were taken at 600 nm every 15 min.

The impact of mechanical pre-treatment on cell cytotoxicity induced by doxorubicin was measured by an MTT colorimetric assay (Merk Life Science S.r.l., Milano, Italia). To examine doxorubicin cell cytotoxicity, cells were seeded on silicone plates at 110 cells/mm² density and allowed to adhere for 24 h as previously indicated. Then, cells on the silicone-plate were 24 h-treated with serum-free medium at different concentrations of doxorubicin within the range of 0–10 μM. For the measurement of absorbance after 24 h, each well received 20 μL of MTT solution (5 mg/mL in PBS with Ca²⁺ and Mg²⁺) and was subsequently incubated at 37°C in a 5% CO₂ atmosphere for 2 h. To dissolve the formazan crystals, 100 μL of extraction buffer (5% SDS in N,N-Dimethylformamide) was then added to each well, followed by another 2 h of incubation at 37°C with 5% CO₂. The absorbance of the formazan products in each well was assessed at a wavelength of 570 nm using the Infinite^® 200 PRO multi-well plate reader from Tecan Group Ltd. in Männedorf, Switzerland after 2 h of incubation. The relative cell viability index was derived by dividing the absorbance at a given doxorubicin concentration over the absorbance recorded for the control cells (which were incubated without doxorubicin).