Ethbr

Ethidium bromide (EthBr) accumulation/efflux and Nile red uptake were measured by fluorescence intensity, as previously described (Chuang et al., 2015 (link)) with minor modifications. Briefly, mid-log-phase or nutrient-starved cultures were washed with PBS and then stained with 2 μg/ml EthBr (Sigma) or with 0.125 μM Nile red (Sigma). For the EthBr accumulation assay with efflux inhibitor, verapamil (Sigma) was added and bacteria incubated with 2 μg/ml EthBr and 100 μg/ml verapamil for 60 min. In all assays, the cells were incubated in 96-well plates, and analysis was performed at the indicated time points by excitation at 544 nm and emission at 590 nm on a FLUOstar OPTIMA microplate reader (BMG LABTECH). All data were normalized to the time zero reading of each well and to bacterial density. All experiments were performed in triplicate and repeated at least twice, yielding similar results.