Luminoskan ascent luminometer varioskan flash

The inner membrane integrity assay was performed as previously described [43 (link),44 (link)]. Briefly, bacteria at an OD₆₀₀ of 1.0 were washed twice with phosphate buffer saline (PBS, pH 7.2) and resuspended to an OD₆₀₀ of 0.5. The bacteria were incubated with 10 μM PI at 25 °C for 30 min. Then the bacterial samples were incubated with or without 0.78 μg/mL polymyxin B at 37 °C for 1 h. The fluorescence values of the samples were detected under the conditions of excitation wavelength 535 nm and emission wavelength 615 nm with a Luminoskan Ascent luminometer (Varioskan Flash, Thermo Scientific, Waltham, MA, USA). The result of relative fluorescence intensity was calculated as (100(F–F₀) / F₀)%. F and F₀ represent fluorescence intensities of samples with polymyxin B and without polymyxin B treatment, respectively.

The outer membrane permeability assay was performed as previously described [41 (link),42 (link)]. Overnight cultures of indicated strains were transferred to fresh LB and grown to an OD₆₀₀ of 1.0. Bacterial cells were washed twice with 5 mM HEPES containing 5 mM glucose (pH 7.0) and resuspended in the HEPES buffer to an OD₆₀₀ of 0.5, followed by incubation with 10 μM NPN at 25 °C for 30 min. Then the bacteria were incubated with or without 0.78 μg/mL polymyxin B at 37 °C for another 30 min. The fluorescence intensities (excitation wavelength: 350 nm, emission wavelength: 420 nm) were measured with a Luminoskan Ascent Luminometer (Varioskan Flash, Thermo Scientific, Waltham, MA, USA). The relative fluorescence intensity was calculated as (100(F – F₀) / F₀)%. F and F₀ represent fluorescence intensities of samples with polymyxin B and without polymyxin B treatment, respectively.