Milli q water mq water

The 2-chlorotrytil resin, O-Benzotriazole-N,N,N,N′-tetramethyl-uronium-hexafluoro-phosphate (HBTU), and Fmoc protected N-trityl-l-histidine and d-phenylalanine were purchased from GL Biochem (Shanghai) Ltd. (Shanghai, China). All solvents were purchased of analytical grade from Merck (Milan, Italy). Drugs and chemicals were from Sigma (Milan, Italy). High purity Milli-Q-water (MQ water) with a resistivity greater than 18 MΩ·cm was obtained from an in-line Millipore RiOs/Origin system (Merck, Milan, Italy). The ¹H-NMR spectra were recorded at 400 MHz, and the ¹³C-NMR spectra were recorded at 100 MHz on a Varian Innova Instrument (Agilent Technologies, Milan, Italy) with chemical shift reported as ppm (in dimethyl sulfoxide (DMSO) with tetramethylsilane as internal standard). ESI-MS spectra were recorded on an Infinity 6120 single quadrupole LC-MS system (Agilent Technologies, Milan, Italy).

After 24 h of experimental administration of antibiotics to the biofilm, the antibiotic solution in the test plate and the PS in the control plate were removed. The wells were rinsed with 100 µl PS. Next, biofilms were detached by vortexing (900 rpm) and sonication (both 5 min), followed by collection of the content of the wells in a sterile tube. The vortexing and sonication step was repeated after the addition of 100 µl PS to each well. The sterile tube was centrifuged (5 min at 3000 × g), and the pellet was resuspended in 1.5 ml of PS. For each treatment, 2 wells of a 24-well plate were filled with 600 µl of the cell suspension. 1.5 µl of a 20 mM PMA solution in dH₂O (Biotium, Inc., California, USA) was added to the first well (final concentration of 50 µM). To the second well, 1.5 µl of MilliQ water (MQ water) (Millipore, Billerica, Massachusetts, USA) was added. The plates were vortexed (5 min, 300 rpm, room temperature) in the dark and exposed to light for 10 min, using a LED-lamp (Dark Reader transilluminator, Clare Chemical Research, Dolores, Colorado, USA) (output wavelength 465–475 nm) (Deschaght et al., 2013 (link)).