Outer and inner membrane depolarization of P. aeruginosa The outer membrane depolarization

GW572016 activity of the recombinant peptides was determined by the 1-N-phenylnaphthylamine (NPN) uptake assay of Loh et al. [34] with intact cells of P. aeruginosa using the Fluorescan Ascent FL microplate fluorometer. P. aeruginosa was grown with agitation to an A600 nm = 0.6 and harvested by centrifugation. The cells were washed in 5 mM HEPES, pH 7.8 and resuspended to an A600 nm of 0.5 in the same buffer. The microtiter plate wells were supplemented with cells (200 μL) and NPN dissolved in acetone was added to a final concentration of 10 μM. Then peptides were added to the desired concentration and the intensity of fluorescence was measured at λex = 355 nm and λem = 444 nm. The cytoplasmic membrane depolarization activity of the peptides HKI-272 cell line was determined as previously described with the membrane potential-sensitive dye DiSC3 PCI-34051 concentration [35]. Briefly, P. aeruginosa was grown at 37°C with agitation to an A600

nm of 0.6 and harvested by centrifugation. The cells were washed in 5 mM HEPES, pH 7.8 and resuspended to an A600 nm of 0.05 in the same buffer containing 20 mM glucose and 100 mM KCl. The cells were first treated with 15 mM EDTA pH 8.0 to permeabilize the outer membrane and allow the dye to reach the cytoplasmic membrane. Then, a stock solution of DiSC3 was added to a final concentration of 0.4 μM, and quenching was allowed to

occur at room temperature. The desired concentration of peptides to be tested was added. Membrane depolarization was monitored with the Fluorescan Ascent FL microplate fluorometer by observing the change in the intensity of fluorescence (λex = 646 nm, λem = 678 nm) after the addition of the peptides. Preparation of large unilamellar vesicles (liposomes) and leakage of calcein Large unilamellar vesicles (liposomes) containing pure phosphatidylglycerol (PG) were prepared according to the previously described procedure [27]. Liposome-entrapped calcein and removal of free calcein by Sephadex G-50 chromatography were carried out essentially as described [65]. For the calcein release assay, 10 μL of liposome suspension Montelukast Sodium were diluted in 10 mM Tris-HCl pH 7.4, 150 mM NaCl buffer (final vol of 100 μL) and incubated for 15 min at room temperature in the presence or absence (negative control) of the indicated peptides at 8 μM or in the presence of 1% Triton X-100 (positive control). The change in the intensity of fluorescence (λex = 485 nm, λem = 527 nm) was monitored with a Fluorescan Ascent FL microplate fluorometer. Confocal microscopy Bacteria were grown at 37°C with agitation in PSB medium to mid-logarithmic phase. Then, the cells were harvested by centrifugation, washed three times with 10 mM sodium phosphate buffer, pH 7.