New target for antibiotics promises treatment for multi-drug resistant superbugs univgroningen NatureComms
. 35 mM phosphate buffer was mixed with 10 mM EDTA and pH was adjusted to pH 7.0. POPG/POPC/POPE lipids in ratios 60:30:10 mol% were pipetted from the stock solutions in chloroform into the glass vial. Chloroform was evaporated using argon stream and let to completely dry out for > 1 h in vacuum. The lipid film was rehydrated using a mixture of 1 mM CoCland 0.8 mM calcein dissolved in 50 mM phosphate buffer, pH 7.0, into the lipid concentration of 100 mg/ml.
On the day of the experiment, the extruded liposomes were washed from the external buffer containing free CoCland calcein. The original buffer was replaced with the 35 mM phosphate buffer with 10 mM EDTA, pH 7.0 using gravity separation column with the internal volume of 12 ml packed with Sephadex G-75. The column was first preequilibrated with the 35 mM phosphate buffer with 10 ml EDTA, pH 7.
The fluorescence measurements were performed with Fluorometer QuantaMaster 40 . The liposomes were diluted to 0.1 mg/ml for the experiments. The calcein was excited at λ= 520 nm was detected in time with both the excitation and emission slits opened to the range of wavelengths ± 5 nm. Background fluorescence was measured for 5 min, then membrane-active agent was added and the increase in the fluorescence intensity from the calcein leaking out of the liposomes was detected for 10 min.
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