Growth curves were generated as described in Vohra & Poxton (2011), and culture supernatants were collected by centrifugation at 13 000 g for 1 min. Supernatants were collected at 8 and 12 h (late exponential phase) and 20 and 24 h (stationary phase). The SLP, flagella and HSP preparations CHIR99021 were visualized on SDS-PAGE gels stained with colloidal Coomassie blue stain G250 (Severn Biotech), and Western blots were performed with rabbit antiserum

prepared against whole UV-killed cells of C. difficile (McCoubrey & Poxton, 2001). The protein concentrations in the preparations were determined using the Bradford reagent (Sigma-Aldrich). The quantities of toxin A and toxin B were determined as described in Vohra & Poxton (2011). Endotoxin contamination in the antigen preparations was determined by an end-point LAL

assay using the Pyrochrome® reagent (Associates of Cape Cod) as per the manufacturer’s instructions. THP-1 cells (European Collection Of Animal Cell selleck inhibitor Cultures, ECACC 88081201) were cultured in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% heat-inactivated foetal bovine serum, 6 mM l-glutamine, 10 mM HEPES with 100 U mL−1 penicillin and 10 μg mL−1 streptomycin (sRPMI) at 37 °C in 5% CO2. Monocytic THP-1 cells at a density of 5 × 105 cells mL−1 were incubated with PMA (Sigma-Aldrich) at 10 and 50 ng mL−1 at 37 °C for 24 h for differentiation into macrophage-like adherent cells. Immunofluorescence analysis was performed on the BD FACSCalibur (BD Biosciences) machine, and differentiation

was confirmed using FITC anti-human CD4 antibody and APC anti-human CD11b antibody (eBioscience) and also visually under a microscope. The data were analysed using the Flowjo 9.0 software. Macrophage-like cells were washed with several washes of prewarmed PBS and subsequently challenged with 100 μL of the C. difficile antigens prepared in sRPMI at concentrations of 5 and 10 μg mL−1. For the challenge with culture supernatants, 100 μL supernatant was added to the macrophage-like cells for 3 h, following which the cells were washed and the culture supernatants were replaced with fresh sRPMI. LPS from E. coli R1 (100 ng mL−1) was used as a control. The optimum times for detection of the different cytokines were determined by repeated collection of supernatants at 4 and 24 h (results acetylcholine not shown), and these were found to be 4 h for TNF-α and 24 h for IL-1β, IL-6, IL-8, IL-10 and IL-12p70. The supernatants were stored at −20 °C until use. In-house ELISAs were developed and standardized for the quantification of TNF-α, IL-1β, IL-6, IL-8, IL-10 and IL-12p70. The details of the antibodies and the amounts used are described in Table 1. From repeated assays, the ELISAs were found to be suitable to detect cytokines in the range of 32 ng mL−1–31.25 pg mL−1. Recombinant proteins used as standards for TNF-α, IL-1β, IL-6, IL-10 and IL-12p70 were obtained from PeproTech and that for IL-8 was obtained from eBiosciences.