EtBr efflux assay

Overnight bacterial cultures were diluted 1:100 in fresh CA-MHB medium and grown at 37 °C to an OD₆₀₀ of 1. The cells were washed three times with 10 mM HEPES buffer and adjusted the OD₆₀₀ = 0.5, followed by the addition of 5 μM EtBr in the presence of D-11 or 40 mg/L CCCP and incubated 30 min at 37 °C. The cell was collected by centrifugation and resuspended in MHB medium containing the indicated concentration of D-11 or 40 mg/L CCCP. Then the fluorescence was monitored at excitation/emission of 525/600 nm every 5 min for 90 min at 37°C with a luminometer (Varioskan Flash; Thermo Scientific). All the tests were performed in triplicate.