Membrane Potential Determination in MRSA

The membrane potential of the study strains was determined using a BacLight bacterial membrane potential kit (Life Technologies, Carlsbad, CA, USA) (17 (link)). In brief, MRSA cultures were adjusted to an OD₆₀₀ of 0.4 in 1 mL of filtered PBS, mixed with 10 μL of 3 mM 3,3′-diethyloxacarbocyanine iodide [DiOC2(3)] fluorescent dye, and incubated at room temperature for 30 min. In parallel, a control with an additional 10 μL of 500 μM proton motive force inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was prepared for each sample. The fluorescent signals were determined at an excitation wavelength of 485 nm and an emission wavelength of 528 nm (green fluorescence) and an excitation wavelength of 485 nm and an emission wavelength of 590 nm (red fluorescence) using a BioTek Synergy 2 microplate reader (BioTek Instruments, Winooski, VT, USA). The ratio between red fluorescence and green fluorescence was calculated to indicate membrane potential (17 (link)).