Membrane Permeabilization Assay

This assay was performed based on a protocol adapted from those previously described in literature.^24,25 Bacteria were grown overnight at 37 °C in LB, diluted 50× in Lysogeny Broth (LB), and then re-grown to an OD₆₀₀ of 0.5. The bacterial suspension was centrifuged for 10 min at 1000g. The bacterial pellet was then resuspended in 5 mM HEPES buffer supplemented with 20 mM glucose to a final OD₆₀₀ concentration of 1.0. The test compounds were serially diluted (25 μL) in triplicate in a black, 1/2 area clear-bottom 96-well plate. Colistin (final concentration 100 μg/mL) was used as the positive control and DMSO (25 μL) was used as the negative control. To ensure no interactions between the compounds and NPN occur, three wells were filled as an additional control with 25 μL of the highest concentration of compound, NPN, and buffer without the presence of bacteria. A 0.5 mM stock of NPN in acetone was prepared which was further diluted to 12.5× in assay buffer. The NPN solution (25 μL) was added to each well. The 1.0 OD₆₀₀ bacterial stock (50 μL) was then added to all appropriate wells. Wells that were to receive no bacteria received assay buffer instead (50 μL). After 60 min, the plate was measured using a Tecan plate reader with λ_ex = 355 ± 20 nm and λ_em = 420 ± 20 nm. The fluorescence values obtained were transformed into NPN uptake percentage using the following equation:

where the observed fluorescence (F_obs) is corrected for background using the negative control (F₀). This value is divided by the positive control corrected for the background (F₁₀₀– F₀) and multiplied by 100% to obtain the percentage NPN uptake: