The cells were cultivated for 4 days in RPMI-1640 containing 10% FCS, 10 mm HEPES buffer and 5·5 × 10−5 m mercaptoethanol (Sigma). At day 4 of culture, cells were pulsed for 18 hr with [3H]thymidine (1 μCi/well; DuPont, AR). Then, cells were harvested using a cell harvester (Perkin-Elmer Inc.) and the amount of [3H]thymidine incorporation was determined in a β-scintillation counter. Intracellular staining for cytokine production was performed after stimulation of co-culture for 24 hr with PMA (10 ng/ml), ionomycin (1 μg/ml)

with or without IL-23p19 (10 μg/ml) (ebiosciences, San Diego, CA) in the presence of brefeldin A for the last 6 hr. Finally, CD4+ IL-17A+ interferon-γ (IFN-γ)+ cells were analysed by flow cytometry. Two or three-colour analysis was performed using flow cytometry, DCs were cultured without or with LPS (1 μg/ml) for 20 min at 37°. After washing, DCs were Roxadustat mouse treated with or without 0·01 μm LTC4 for 30 min at 37° in complete medium

in the presence of brefeldin A (5 μg/ml). After 18 hr, cells were fixed in 4% paraformaldehyde and permeabilized with saponin (0·1% in PBS). The permeabilized cells were incubated with a PE-conjugated anti-IL-12p40 antibody (BD Pharmingen, Trento, NJ) in PBS 0·5% BSA or similarly labelled isotype-matched control antibodies for 30 min. The stained cells were washed with saponin buffer twice and resuspended in isoflow. In some cases, intracytoplasmic cytokines were evaluated in co-cultures of mixed lymphocyte reaction (MLR) and permeabilized cells were incubated with PE-conjugated anti-IL17A AZD6244 molecular weight and FITC-conjugated anti-IFN-γ antibodies (BD Pharmingen). In all cases, the surface staining with FITC-conjugated anti-CD11c (DCs) or Peridinin chlorophyll protein-conjugated Ergoloid anti-CD4 antibodies (BD Pharmingen) was performed before to permeabilization. The staining was analysed by flow cytometry on FACS using cellquest software (BD Biosciences, San Jose, CA). The cytokine levels in supernatants of DCs were measured by ELISA. Assays for IL-12p70, p40, IL-23, IL-6, tumour necrosis factor-α (TNF-α), IFN-γ (eBiosciences) and IL-17 (Quantikine; R&D Systems, Bs. AS, AR) were performed according

to the manufacturer’s protocols. The limits of detection were: 15 pg/ml (IL-12p70; p35/p40), 30 pg/ml (IL-23; p19/p40), 10 pg/ml (IL-12p40), 8 pg/ml (TNF-α), 4 pg/ml (IL-6), 15 pg/ml (IFN-γ) and 5 pg/ml (IL-17). The significance between means was assessed by Student’s paired t-test. P ≤ 0·05 was determined to indicate statistical significance. We decided to evaluate whether LTC4 is able to modulate the central molecules expressed by DCs that are involved in the activation of T lymphocytes.3,4 In the first place, we studied the concentrations of LTC4 able to modulate the expression of the MHC class II molecules. To analyse this point, DCs were cultured in the presence of different concentrations of LTC4 (10−5–10−9 m) at 37°. After 18 hr, its expression was analysed by flow cytometry.