4.8. Outer Membrane Permeability Assay

The fluorescent probe NPN was used to evaluate the outer membrane integrity of E. coli B2 when treated by different concentrations of equisetin combined with colistin or with drugs alone. Briefly, E. coli B2 grown overnight at 37 °C were washed and resuspended in 5 mmol/L of HEPES (pH 7.2, +5 mmol/L of glucose) to obtain an OD₆₀₀ of 0.5. NPN was added to a final concentration of 10 µmol/L and the mixture was incubated at 37 °C for 30 min. A total of 190 µL of probe-labelled bacterial cells were added to a 96-well plate and then 10 µL of the drug combination or the drug alone was added. After incubating for 30 min, fluorescence was measured on an Infinite M200 Microplate reader (Tecan) with the excitation wavelength at 350 nm and the emission wavelength at 420 nm.