L. monocytogenes strains were grown to mid-late-logarithmic phase in BHI media at 37°C. Bacteria were diluted in phosphate-buffered saline (PBS) to a concentration of 106/ml and transferred to a fluorescence-activated cell sorter (FACS) tube. Next, cells were stained with 3 mM membrane potential indicator dye DIO2(3) (3,3′-diethyloxacarbocyanine iodide) (Sigma; 320684) and/or 500 µM proton ionophore CCCP (carbonyl cyanide 3-chlorophenylhydrazone) (Sigma; C2759) for 30 min. Samples were analyzed on a BD LSR-II flow cytometer, and data analysis was performed using FlowJo software. Samples were gated for bacteria using forward and side scatter, and then individual bacteria were measured for ratiometric fluorescence analysis using comparisons between red mean fluorescence intensity and green mean fluorescence intensity.