Fluorescence associated with washed, solubilized cells was quantitated and correlated to vesicle amount using standard curves generated for vesicles from each strain. Experiments were done in triplicate, SEM www.selleckchem.com/products/th-302.html is indicated for 2 to 7 separate experiments. At the 24 h time point, p < 0.001 for each data set. To test whether the vesicles would interact

similarly with primary cells, we incubated vesicles with human bronchial epithelial (HBE) cells from healthy human volunteers (Fig. 1B). The results for the HBE cells were similar to those with cultured cells, thus cultured cells appeared to be a good model for primary cells in further assays. Together, these data indicate that P. aeruginosa vesicles from CF strains associate to a greater extent with epithelial cells than vesicles from a non-CF strain. When we tested temperature dependence of vesicle-host cell association we found that incubation at 4°C substantially decreased the amount of S470 vesicles associated with the lung cells, whereas

little-to-no difference was observed for PAO1 vesicles (Fig. 2A). These data indicate that a temperature-dependent mechanism was responsible for the differences observed in the association between vesicles from a CF strain and vesicles from a non-CF Staurosporine mouse strain. Figure 2 S470 vesicle association with host cells is temperature-dependent. A, FITC-labeled-vesicles (2.5 Metformin μg per well) were incubated with A549 cells (5 × 104 cells per well) for 24 h at 37°C (black bars), or 4°C (gray bars). SEM is indicated, n≥2, in triplicate.

B and C, A549 cells alone (left panels) or incubated with 2.5 μg FITC-labeled S470 vesicles (green, right panels) for 6 h at 37°C (B) or 4°C (C). After incubation, cells were washed, labeled with AF633-WGA (blue), fixed in 2% paraformaldehyde, and visualized by confocal microscopy. Pseudomonas aeruginosa vesicles are trafficked into lung epithelial cells Temperature-dependent association of S470 vesicles suggested that these vesicles may be find more internalized by the lung epithelial cells. We used confocal microscopy to analyze vesicle-host cell interactions. Cultured A549 cells were incubated with FITC-labeled S470 vesicles for 6 hours at 37°C, and plasma membranes were stained with AF633-wheat germ agglutinin (WGA) to visualize cell boundaries. At 37°C, vesicle fluorescence appeared to be mostly internal and concentrated in a perinuclear region of the cell (Fig. 2B). Very little vesicle association was observed for incubations maintained at 4°C (Fig. 2C). Thus, both binding and internal localization of S470 vesicles was affected at the lower temperature. To further confirm vesicle internalization, vesicles were labeled using AF488 instead of FITC to maximize fluorescence and minimize the effects of photobleaching.