Cells were gated on forward scatter and side scatter to exclude clumps and debris. DCs were CD14- and DC-SIGN+ (constituting approximately 90%

of gated cells). Results were analysed using Summit software version 4.3 (Dako/Beckman Coulter). Cytokine Analysis Dendritic cells were infected with live H37Ra or streptomycin-killed H37Ra at MOI 1 for 24 or 48 h. LPS was applied for 24 h (Sigma; 1 μg/ml) as a positive control for DC maturation and cytokine secretion. Cytokine secretion was measured in cell-free supernatants by ELISA using the Meso Scale Discovery SECTOR Imager 2400 and the following assays: human IL-6 assay, and human Th1/Th2 10-cytokine multiplex assay, capable of detecting IFN-γ, IL-1β, IL-10, IL-12p70, IL-13, IL-2, IL-4, IL-5, IL-8 and TNF-α (Meso Scale Discovery, Gaithersburg, MD). IL-4 measurements were disregarded, as DCs were maintained in culture with exogenous IL-4, rendering it impossible to distinguish levels

check details secreted by the cells themselves. Colony forming units and BacT/ALERT 3D Dendritic cells were harvested 24 h or 72 h after infection with M. tuberculosis. VEGFR inhibitor Cells were centrifuged and washed 3 times at 800 rpm to remove extracellular bacteria. The cells were lysed in 0.1% Triton X-100 (Sigma) for 10 min. The resultant bacterial suspension was then passed through a 25 gauge needle eight times to disperse clumps. The bacilli were serially diluted x10-1 – x10-5 in Middlebrook 7H9 medium and plated on Middlebrook 7H10 agar (Difco) supplemented with oleic acid-albumin-dextrose-catalase (Becton Dickinson) and cycloheximide (Sigma), or inoculated into BacT/ALERT MP bottles (bioMérieux, Durham, NC). Agar plates were incubated at 37°C for 14 – 21 days and colony forming units were counted. BacT/ALERT MP bottles were incubated in a BacT/ALERT 3D automated microbial detection system (bioMérieux) and time to reach positivity was recorded, and a growth index was calculated, using the equation ((TTPDay1-TTPDay3)/TTPDay1)x100 as we have already published for the BD BACTEC

liquid culture platform [69]. 4��8C In this equation TTP Day 1 is the time to culture positivity for infected DC lysates at Day 1, and TTP Day 3 is the time to positivity for infected DC lysates at Day 3. Statistical analysis Results are expressed as means ± the standard errors of the mean (SEM). The data were analyzed with GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA) statistical software using by repeated measures ANOVA with Tukey’s post test, or (where stated) by the Friedman test followed by Dunns multiple comparison test. A P value of < 0.05 was considered statistically significant. Graphs were compiled using GraphPad Prism 5 software. Acknowledgements The authors wish to thank Dr Timothy Grant, Centre for Support and Analysis in Research (CSTAR) for providing advise on statistical analysis of the data.