Propidium iodide

The cell membrane permeability was tested with propidium iodide (J&K Scientific Ltd.) dye as described^{58 (link)}. In brief, the overnight culture of Staphylococcus aureus ATCC 6538 was harvested and washed twice using buffer A (5 mM HEPES buffer, 5 mM glucose, pH 7.0). The cells were resuspended in the same buffer and adjusted to an optical density of 0.5. Subsequently, the aliquots of cell suspension (1 mL) were treated with ART B or ART 9B at 37 °C for 1 h. After propidium iodide (20 μM) was added, the cells were incubated for 20 min at 37 °C in dark, harvested by centrifugation, washed once, and resuspended in 1 mL buffer A for measuring fluorescence intensity spectrophotometrically in 96-well plate (at an excitation wavelength of 535 nm and an emission wavelength of 615 nm). Solvent DMSO was employed as a negative control; daptomycin (6 μM) + CaCl₂ (5 mM) was employed as a positive control.