Methods Chemicals and antibodies RPMI-1640 medium containing 1 mM sodium pyruvate, Dulbecco’s phosphate-buffered saline (D-PBS) and Hanks’ balanced salt solution (HBSS) were PXD101 purchased from Gibco (Scotland). Middlebrook OADC (oleic acid albumin dextrose catalase) enrichment, Middlebrook 7H9 broth, and Middlebrook 7H10 agar were obtained from Becton Dickinson (USA). IFN-γ, phorbol 12-myristate 13-acetate (PMA), bovine serum albumin (BSA), fluorescein isothiocyanate (FITC), Tween-20, Tween-80, IRAK1/4 inhibitor, 37% formaldehyde solution (FA), horseradish
peroxidase (HRP), 2-mercaptoethanol NVP-HSP990 (2-ME) and luminol were purchased from Sigma-Aldrich (USA). Human type AB serum (off-clot) and fetal bovine serum (FBS) were purchased from PAA-The Cell Culture Company (Austria). Mouse IgG2a anti-human TLR2 (sodium azide-free), phycoerythrin (PE)-conjugated mouse anti-TLR2 (IgG2a), and PE-conjugated mouse IgG2aκ isotype control were obtained from Imgenex (USA). FITC-conjugated mouse anti-human CD14 (IgG2aκ) and PE-conjugated anti-human CD11b (IgG1κ) were purchased AZD9291 purchase from BD Pharmingen (USA). Human TNF-α and human IL-10 Quantikine enzyme-linked immunosorbent assay (ELISA)
kits were purchased from R&D Systems (USA). Bacterial strains and growth conditions All strains used in this study were based on M. tuberculosis H37Rv (ATCC) and were maintained on Middlebrook 7H10 agar or 7H9 broth supplemented with 10% OADC enrichment and 25 μg/ml kanamycin, as required. For growth on media supplemented with defined carbon sources, strains were grown
in minimal medium supplemented with 0.01% cholesterol, as described previously . The engineering of the Mtb strain deficient for the KstD enzyme (ΔkstD), and ΔkstD complemented with an intact kstD gene (ΔkstD-kstD) was described previously . Wild-type, mutant, and complemented bacterial strains were prepared for infection by growing in roller bottles in Middlebrook 7H9 broth containing 10% OADC enrichment and 0.05% Tween-80 for 4–6 days to reach an optical density at 600 nm (OD600) of 1. A portion of the bacterial culture (approximately 1 × 109 bacilli/ml) Ureohydrolase was suspended in Middlebrook 7H9 broth and labeled with 100 μg/ml of FITC by incubating for 2 hours at room temperature with gentle agitation in the dark. FITC-labeled bacteria were washed once with Middlebrook 7H9 broth supplemented with 4% BSA and then twice with Middlebrook 7H9 broth without BSA. Unlabeled and FITC-labeled bacteria were divided into equal portions and stored at -85°C. After 1 week, a portion of bacteria was thawed and colony-forming assays were used to determine the number of bacterial colony-forming units (CFUs).