For use in mouse pups, optimal intranasal exposure volumes for lung deposition were determined. Volumes of 5-, 10- or 15-μl Evans Blue solution were deposited on the nostrils under isoflurane anaesthesia. After 15 min, the pups were killed by cervical dislocation. The 10-μl volume was determined to give maximal lung deposition by visual inspection of the blue-colouring

of the lungs and stomach. Selection of 30 μl as the booster volume (Table 1) was based on the estimated 300% increase in body mass from 1 to 4 weeks of age [16, 17]. Determination of OVA-specific antibodies in serum.  OVA-specific IgE antibodies were detected in a capture ELISA as previously described by Lovik et al. [18] and modified by Ormstad et al. [19]. Poly-HRP-streptavidin (Thermo-Scientific,

Pierce Biotechnology Inc., Rockford, IL, USA) followed by Stabilized chromogen TBM (Invitrogen, Camarillo, CA, USA) was used for detection and the reaction Hydroxychloroquine cost stopped with 2 N H2SO4 solution. OVA-specific IgG1 was measured in a capture ELISA as previously described [13]. The sera to be tested were analysed in duplicates following optimal dilution; 1:50 for IgE and 1:200 or 1:200,000 for IgG1. For both antibody assays, a standard curve was included on each plate. Standard curves were NVP-BKM120 datasheet made from duplicates of diluted IgE standard (mouse anti-OVA IgE, AbD Serotec) or serum pools from mice immunized with OVA and Al(OH)3 for IgG1. OD was measured at 450 nm on a MRX Microplate Reader (Dynatech Laboratories, Chantilly, MTMR9 VA, USA) connected to a PC using Revelation software (Thermo Labsystems, Chantilly, VA, USA). Lymph node preparation and determination of cytokine release.  Single-cell suspension of SLNs and MLNs was prepared

by forcing the lymph nodes through a 70-μm cell strainer (BD Labware, Franklin Lakes, NJ, USA). The cells were washed and then counted in a Coulter Counter Z1. After incubation in culture medium (RPMI 1640 with 10% foetal calf serum, 100 U penicillin G, and 0.1 mg/ml streptomycin) with or without 1 mg/ml OVA at 37 °C and 5% CO2 for 4 or 5 days (differed for practical reasons between the i.n. and i.p. study, respectively), the supernatants were removed and stored at −20 °C until cytokine measurements. The levels of IL-4, IL-5, IL-10, IL-13, IFN-γ, and in the i.n. study also IL-17, were determined using BD CBA Mouse Soluble Protein Flex Sets measured on a BD LSR II flowcytometer and analysed by the FCAP Array software (all from BD Biosciences, San Jose, CA, USA) according to the manufacturer’s protocol. Assessment of inflammatory cells and cytokines in BALF.  Cells in BALF were prepared and stained with the Hemacolor rapid staining of blood smear kit (Merck KGaA, Darmstad, Germany) as previously described [13]. Cell differential counting of blinded slides was performed by microscopic examination by the same investigator (JSH). In the i.n.