respective suppliers using standard protocols. S9 and IB3-1 cells were cultured in basal LHC-8 (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, and 1 mM sodium pyruvate. NuLi-1/CuFi-1 cells were cultured in BEBM serum-free medium (Lonza) with supplement bullet kit (EGF, hydrocortisone, bovine pituitary extract, transferrin, bovine insulin, triiodothyronine, epinephrine, retinoic acid), 2 mM L- glutamine, and 1 mM sodium pyruvate. PBMCs from CF patients and controls were cultured in RPMI-1640 (Hyclone) supplement- ed with 10% FBS, 2 mM L-glutamine, and 1 mM sodium pyruvate (complete RPMI). THP1-XBlue cells were cultured in complete RPMI with the addition of zeocin (100 mg/ml) to select for cells expressing the SEAP NF-kB/AP-1 reporter. Prior to stimulation, bronchial epithelial cell lines were plated in coated [62] 96-well plates (BD Biosciences) at 36104 cells/well unless indicated, and allowed to adhere overnight. Plates for S9 and IB3- 1 stimulations were coated in a mixture of bovine serum albumin (100 mg/ml), fibronectin (10 mg/ml), and bovine collage type I (30 mg/ml) (BD Biosciences). Plates for NuLi-1 and CuFi-1 were coated with collagen type IV (60 mg/ml) (Sigma Aldrich). PBMCs were plated in 96-well plates at a density of 1.56105 cells/well in 200 ml (7.56105 cells/mL). THP-1 reporter cells were differenti- ated into a macrophage-like phenotype using 50 ng/ml of phorbol 12-myristate 13-acetate (PMA) (Sigma Aldrich) for 24 hours at a density of 16105 cells/well in 200 ml (56105 cells/ml). Cells were washed with PBS and allowed to rest a further 42 hours prior to stimulation. CF and control subject PBMCs The diagnosis of CF was established by typical clinical features, increased sweat chloride concentrations (.60 mmol/l), and detection of CF-inducing mutations. All patients with CF were clinically stable at the time of blood donation, and we excluded any subjects who were receiving systemic corticosteroids due to potential immunomodulatory activity. Control samples were provided by healthy adult volunteers. In previously published work we have demonstrated that TLR-mediated inflammatory responses are stable in humans from birth to 60 years old [63], therefore did not age-match the CF patients and control subjects. Peripheral blood was collected in sodium heparin tubes (BD Biosciences) and PBMCs were isolated using density gradient centrifugation on Ficoll-PaqueTM Plus (GE Healthcare). The layer containing PBMCs was isolated, washed twice in PBS and resuspended in complete RPMI. Cells were enumerated by trypan blue exclusion using the Countess automated cell counter (Invitrogen). For derivation of macrophages from monocytes, monocytes were allowed to adhere to plastic for 2 hours in RPMI 1640 after which non-adherent cells were removed. Monocytes were allowed to differentiate in RPMI 1640 supplemented with 10% human AB serum for 10 days. Cell stimulation and cytokine quantification Bronchial epithelial cells were plated and allowed to adhere overnight prior to stimulation. Bronchial epithelial cells were rested or primed with LPS for 5 hours and stimulated with live P. aeruginosa PAO1 or ATP for the times indicated. PBMCs and THP-1 reporter cells were either rested or primed with LPS (Invivogen) or heat-killed PAO1 overnight (16 hours). The next day the cells were challenged with live PAO1, PAO1DexsA, ATP (Invivogen), or Poly(dA:dT) (Sigma Aldrich) for the times indicated (see Fig. 1). For stimulations with Poly(dA:dT), lipofectamine LTX was used at a 1:1 (w:v) ratio of mg of DNA to ml of lipofectamine and was mixed 30 minutes prior to stimulation. The NF-kB inhibitor Bay11-7082 (Invivogen) was added to cultures 1 hour IL-1b Production in Cystic Fibrosis prior to priming. If no priming was involved, inhibitor was added 1 hour prior to inflammasome stimulation. The CFTR inhibitor CFTRinh172 (Sigma Aldrich) was added to cultures 18 hours prior to inflammasome stimulation. The caspase-1 inhibitor z-YVAD- fmk (Biovision) was added to cultures 1 hour prior to inflamma- some stimulation. Supernatants were collected and stored at 220uC. Cytokines released into supernatants from PBMCs stimulated with inflammasome activators were quantified using sandwich ELISA (eBioscience). Immunoblotting 16106 cells were seeded in 12-well plates, stimulated as indicated, and lysed in RIPA buffer supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Scientific). Protein concentrations were determined by Bradford assay (Thermo Scientific). Lysates were resolved by electrophoreses on 10% SDS-polyacrylamide gels and transferred onto PVDF membranes (Millipore). Blots were blocked for 1 hour at room temperature and probed overnight at 4uC for pro-IL-1b (Santa Cruz), IkBa (Cell Signaling), or b-actin (Cell Signaling). Blots were subsequently probed with fluorescently-labeled secondary anti- bodies, IRDyeH 680 or 800CW (LI-COR Biosciences) for 1 hour. Both blocking and probing steps were carried out in tris-buffered saline (G Biosciences) containing 5% bovine serum albumin and 0.1% TWEEN 20 (Calbiochem). Blots were imaged on a LI-COR Odyssey infrared imaging system (LI-COR Biosciences) and quantified using the included analysis software. Quantification of caspase-1 activity Bronchial epithelial cells were plated in 6-well plates at 56105 cells/well overnight. Cells were primed with LPS for 5 hours and stimulated with ATP for 1 hour or stimulated with live PAO1 for 3 hours. PBMCs were stimulated the same day as blood donation. PBMCs were seeded in a 96-well plate at a density of 4.56105 cells/well (2.56106 cells/ml), primed with LPS for 5 hours and then stimulated with ATP for 1 hour or Poly(dA:dT) for 3 hours or stimulated with live PAO1 for 3 hours. Caspase-1 activity was measured using FLICA (Immunochemistry Technologies), a cell- permeable fluorescent probe (FAM-YVAD-fmk) that binds active caspase-1. Cells were incubated 1 hour with FLICA at 37uC and stained with PE-Cy7-conjugated anti-CD14 antibodies (eBioscience) to identify monocytes. The gating strategy consisted of including live cells that were CD14 positive which were subsequently analyzed for the frequency of FLICA positive cells. NF-kB/AP-1 Activity Assay Supernatants from THP-1 reporter cells were incubated with Quanti-Blue substrate (Invivogen) at 37uC and allowed to develop for 16–18 hours. Quanti-Blue contains a substrate for alkaline phosphatase and changes in the amount of NF-kB/AP-1 activity were quantified by optical density (l = 655) measured using a SpectraMax 384 Plus plate reader and SoftMax Pro software (Molecular Devices). Bacterial strains P. aeruginosa laboratory strains PAO1 and the PAO1DexsA mutant were obtained from Dr. Robert Hancock. P. aeruginosa strains PAO1 and PAO1DexsA were grown from overnight cultures in Luria Bertani (LB) broth and LB+streptomycin (150 mg/ml) until mid-logarithmic phase. Cells were washed once in PBS and resuspended in PBS to an optical density of 0.5 (l = 600 nm). To prepare heat-killed bacteria, live PAO1 was resuspended in PBS to an optical density of 0.5 and heated at 60uC PLoS ONE | www.plosone.org 10 May 2012 | Volume 7 | Issue 5 | e37689