Animals.  C3H/HeN mice (Charles River Ltd, Margete, UK) 6–9 weeks of age of both genders were used in these studies. The animals were maintained at the animal premises of Ullevål University Hospital, Oslo, Norway. The experiments were approved by the Norwegian Ethics Committee for Animal Research and performed according to the NIH guidelines for PLX4032 solubility dmso the use of experimental animals. Antigen. 

The hapten oxazolone (OXA, [4-ethoxymethalylene-2-phenyl-2-oxazolin-5-one]) was purchased from Sigma (St Louis, MO, USA). Sensitization and elicitation of CS.  Mice were sensitized and elicited according to a variation of an oral mucosa CS model [10]. Briefly, for sensitization 20 μl of 1% OXA in acetone/olive oil (1/10, v/v) was applied once on both sides of the ears or the inner face of the cheeks. One week later, animals were challenged with 10 μl of 1% OXA, topically applied onto both sides of both ears and on the mucosal surface of both cheeks with a total exposure of 60 μl. Sensitized and elicited as well as control mice exposed only once to the hapten were sacrificed at 0,

4, 6, 8, 12, 24, 48, 72 and 168 h after first or second hapten exposure in line with protocols published previously [8, 10]. The experimental series relating to cytokine measurements were performed thrice, and the graphs demonstrated represents typical results from one series of experiments. The experimental series demonstrating weight of lymph nodes and counting of lymph node cells (vide infra) are based upon 4–6 and two individual observations, PXD101 clinical trial respectively. Specimen treatment and ELISA analyses.  Buccal mucosa and ear skin as well as lymph nodes, i.e. regional (two submandibular and two auricular) and distant (four axillary) were excised from both sides of the mice. The buccal mucosa specimens were trimmed to a thin sheet of lamina propria and

epithelium. The ears were split along the cartilage, and specimens containing epidermis and dermis Tideglusib were harvested. All specimens were weighed and immersed separately in 200 μl phosphate-buffered saline (PBS), pH 7.4. The PBS contained 1% bovine serum albumin, 0.5 m EDTA, 2% soy bean trypsin inhibitor and 2% phenylmethylsulphonylfluoride according to the method described by Villavedra et al. [20]. The specimens were frozen at −70 °C until further processed and analysed for cytokines. After thawing, saponin (2%) was added to the specimens and kept in cold (4 °C) overnight. After whirl mixing and centrifugation (1500 g for 5 min), the supernatants were collected and analysed with respect to IL-2 and IFN-γ, using BD™ OptEIA ELISA Sets (Pharmingen; BD™ Biosciences, San Diego, CA, USA). The biotinylated secondary Ab with streptavidin containing horse-radish peroxidase was developed by hydrogen peroxide and TMB (3, 3′, 5, 5′ tetramethylbenzidine). The reaction was stopped using 1 m sulphuric acid.