The tested bacterial strains were grown anaerobically
to mid-exponential phase and then harvested by centrifugation SCH727965 concentration prior to infect the monolayers in 96-well microtiter plates at a this website multiplicity of infection of 100:1. After incubation of 1 h to allow bacterial entry into the cells, monolayers were washed twice with phosphate-buffered saline (PBS), and 100 μL of RPMI containing gentamicin (200 μg × ml-1) was added to each well. The plates were then incubated for 2 h to kill any remaining extracellular bacteria. In the case of the strains carrying vectors, the medium was supplemented additionally with chloramphenicol during the entire assay. The medium was removed and cells were washed twice with PBS. Then, the cells were lysed with sodium deoxycholate (0.5% w/v, in PBS). The number of intracellular bacteria (CFU at t3) was determined plating onto LB agar plates with chloramphenicol (the strains carrying plasmid) or without antibiotic (the wild type strains). Quantitative invasion assay values were calculated as follows: Statistics All results are expressed S63845 in vitro as means ± SD of an individual experiment performed in triplicate. P values were calculated according to Student’s t-test, and values p < 0.05 or p < 0.01 were considered statistically significant. Acknowledgements UNAB Grant DI-05/I (A.T) and FONDECYT Grant 1060999 (G.M). References
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