The infected cultures were incubated at 37°C and 5% CO2 for intended durations. For immunofluorescence, cells were grown on coverslips. Infected cells 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| harvested LBH589 mouse by rubber scraper at different time points as per experimental protocol. The cell pellets for PCR/reinfection as well as supernatants for cytokine analysis were stored at −80°C. Mock infected controls were prepared for every set of experiment to assess the contribution of host cell debris. Control samples were routinely checked for the presence of chlamydia antigens in the donor samples by immunofluorescence microscopy.

Immunofluorescence microscopy The infected monocytes and DCs after intended incubation were fixed in 2% para-formaldehyde for 10 min and washed 3 times in PBS. Cells were permeabilized with 0.5% TritonX-100 for 3 minutes. Following fixation, the cells were blocked with PBS containing 1% BSA and 1% FCS. Genus-specific fluorescein isothiocyanate (FITC)-labelled monoclonal antibody (Pathfinder Chlamydia Confirmation System; Bio-Rad, Redmond, WA) was used to stain the chlamydial inclusions, while the monocytes and DCs were counterstained with Evan’s Blue at room temperature for 45 min. The samples were then washed once with PBS and then washed twice with PBS/DAPI (1:2500) to stain the cell nuclei. Images were

captured in Vistusertib cost 10 random fields with a fluorescence microscope (Leica DMLB, Germany) with standard filters at 63X magnification. ImageJ was used to count the number of inclusions/cells in replicate samples. Data from 3 independent experiments were combined to calculate the mean and standard deviation. Analysis of the infectivity of C. trachomatis in monocytes/DCs Cells

harvested at different time points were lysed in an ultrasonic sonicator bath (Jürgen’s Hannover, Germany). Cell lysates were used to infect HeLa cells seeded on coverslips and cultured in MEM media containing 1 μg/ml cycloheximide at 37°C in 5% CO2 for the intended duration. At the end of the infection period, cells were fixed for 10 min in absolute methanol, air-dried, and stained using FITC-labelled monoclonal antibody (Pathfinder Chlamydia Confirmation System; Bio-Rad, Redmond, WA) and counterstained with Evan’s blue. Images were captured in 10 random fields with a fluorescence microscope (Leica DMLB, Germany) with Protirelin standard filters at 40X magnification. The inclusions were counted as described under section Immunofluorescence microscopy. Data from 3 independent experiments were combined to calculate the mean and standard deviation. Gene expression analysis by real-time PCR For the analysis of chlamydial gene expression, infected cells were harvested at different time points and real-time PCR was performed targeting the 16S rRNA gene as described previously [34]. To analyse chlamydial developmental phase, expression of genes euo, ompA and omcB were performed.