Membrane depolarization assay using DiSC3(5) fluorescent dye

To determine whether bovine NK-lysin peptides have the ability to disrupt electrochemical potential across the M. bovis membrane, a cytoplasmic membrane depolarization assay was performed using a membrane potential-sensitive cyanine dye 3,3'-dipropylthiadicarbocyanine iodide (DiSC₃(5); AnaSpec, Fremont, CA) as described previously, but with some modifications [34–36]. Briefly, M. bovis was harvested from overnight cultures by centrifugation (10,000 × g for 10 min) and washed once with 5 mM HEPES-20 mM glucose buffer, pH 7.4. Mycoplasma bovis (~1 × 10⁸ CFUs/ml) was resuspended in the same buffer, DiSC₃(5) (0.4 μM final concentration) was added, gently mixed by pipetting, and 100 μl aliquots (~1 × 10⁷ CFUs) were transferred into a 96-well opaque plate and incubated at 37˚C in a humidified atmosphere of 5% CO₂ to obtain stable reduction of DiSC₃(5) fluorescence (~30 min). To equilibrate cytoplasmic and external K⁺ concentrations, 100 μl of 200 mM KCl, pH 7.4 was added into each well and incubated for another 10 min. NK-lysin peptides (5 and 20 μM final concentrations) were added and changes in fluorescence were immediately measured using a fluorescent microplate reader with excitation and emission wavelengths at 622 nm and 670 nm, respectively. Triton X-100 (0.1% (v/v) final concentration) treated M. bovis was used as a positive control, while ethanol treated (killed) and untreated M. bovis (in PBS) were used as negative controls.