LPS presence was determined by measuring the 3-deoxy-d-manno-2-octulosonic acid (Kdo) content by the thiobarbituric acid method modified to correct interference due to deoxysugars [22]. Kdo content was less than 0.07%. Mammalian cell culture and bacterial infection Monolayers of human

lung carcinoma cells (A549, ATCC CCL185) derived from type II pneumocytes were grown to confluence as described before [13]. Cells were serum starved for 18 h before infection. Overnight-grown bacteria were {Selleck Anti-infection Compound Library|Selleck Antiinfection Compound Library|Selleck Anti-infection Compound Library|Selleck Antiinfection Compound Library|Selleckchem Anti-infection Compound Library|Selleckchem Antiinfection Compound Library|Selleckchem Anti-infection Compound Library|Selleckchem Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|buy Anti-infection Compound Library|Anti-infection Compound Library ic50|Anti-infection Compound Library price|Anti-infection Compound Library cost|Anti-infection Compound Library solubility dmso|Anti-infection Compound Library purchase|Anti-infection Compound Library manufacturer|Anti-infection Compound Library research buy|Anti-infection Compound Library order|Anti-infection Compound Library mouse|Anti-infection Compound Library chemical structure|Anti-infection Compound Library mw|Anti-infection Compound Library molecular weight|Anti-infection Compound Library datasheet|Anti-infection Compound Library supplier|Anti-infection Compound Library in vitro|Anti-infection Compound Library cell line|Anti-infection Compound Library concentration|Anti-infection Compound Library nmr|Anti-infection Compound Library in vivo|Anti-infection Compound Library clinical trial|Anti-infection Compound Library cell assay|Anti-infection Compound Library screening|Anti-infection Compound Library high throughput|buy Antiinfection Compound Library|Antiinfection Compound Library ic50|Antiinfection Compound Library price|Antiinfection Compound Library cost|Antiinfection Compound Library solubility dmso|Antiinfection Compound Library purchase|Antiinfection Compound Library manufacturer|Antiinfection Compound Library research buy|Antiinfection Compound Library order|Antiinfection Compound Library chemical structure|Antiinfection Compound Library datasheet|Antiinfection Compound Library supplier|Antiinfection Compound Library in vitro|Antiinfection Compound Library cell line|Antiinfection Compound Library concentration|Antiinfection Compound Library clinical trial|Antiinfection Compound Library cell assay|Antiinfection Compound Library screening|Antiinfection Compound Library high throughput|Anti-infection Compound high throughput screening| subcultured and grown to exponential phase, harvested by centrifugation (20 min/2700 × g) and resuspended in PBS. The inoculum for the infection was prepared in Earle’s buffered salt solution (EBSS), pH 7.4. A549 cells (80–90% confluent) seeded on glass coverslips in 24-well tissue culture plates were subsequently infected with K. pneumoniae strains at a multiplicity of infection (MOI) ranging from 100:1 to 1000:1 and centrifuged for 4 min at 200 × g at 22°C. Infected plates were then incubated for 2 to 5 h at 37°C/5% CO2 in a humidified LBH589 incubator. For adhesion

assays, cells were washed five times with 1 ml phosphate-buffered selleck chemicals llc saline (PBS) pH 7.4 after 2 h of infection and lysed with 0.5%-Triton in PBS. Serial dilutions of the lysates in PBS were plated on LB plates for quantification of viable bacteria. Experiments were carried out in triplicate in three independent occasions and results are expressed as % adhesion = 100 × (n° of bacteria recovered from well/initial n° of bacteria added). Where indicated, bacteria were UV killed by exposure to 1 joule for 3 min in a BIO-LINK BLX crosslinker (Vilber Lourmat). Fluorescence microscopy Cell monolayers were fixed in 3.7% paraformaldehyde in PBS. Rhodamine (RRX)-conjugated phalloidin (Molecular

Probes) diluted 1:200 in 10% horse serum/0.1% saponin in PBS was used to stain the actin cytoskeleton. Coverslips were washed twice in PBS containing 0.1% saponin, once in PBS, and incubated for 30 min with phalloidin-RRX. The coverslips were then washed twice in 0.1% saponin in PBS, once in PBS and once in H2O, mounted in Aqua-Poly/Mount (Polysciences) Protirelin and analysed with a Leica CTR6000 fluorescence microscope. Analysis of host cell DNA integrity after K. pneumoniae infection A549 cells were infected with K. pneumoniae strains at MOI of 500:1 in tissue culture plates. 6 h post-infection, cells (~2.5 × 106) from 2 wells were collected in PBS by scraping and lysed in 600 μl cold lysis buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.1% SDS). Proteinase K (100 μg/ml) was added and samples were incubated for 3 h at 55°C. Samples were cooled to 22°C and incubated with 20 μg/ml RNase (DNase-free) for 20 min at 37°C. 200 μl 5 M potassium acetate were added and samples were centrifuged (13000 × rpm, 22°C, 1 min). DNA present in the supernatants was precipitated with isopropanol, washed in 70% ethanol and dissolved in sterile water.