This regimen stimulates the development of Th1-polarized immunity to OVA. On day 50, the mice were anaesthetized and challenged with 100 µg of OVA in 30 µl of Palbociclib research buy PBS by footpad injection. The DTH reaction was assessed by measuring tissue swelling in the footpad after 24 h (i.e. on day 51) using a caliper; the mice were then killed. To measure sensitization, the popliteal lymph nodes were excised and single cells were prepared under aseptic conditions and suspended in Iscove’s
medium supplemented with 2 mM l-glutamine, 50 µM mercaptoethanol, 50 µg/ml gentamycin and 10% fetal calf serum (all from Sigma, Steinheim, Germany). Samples of 1 × 105 cells/well were transferred to 96-well microtitre plates and stimulated with 0·5 mg/ml OVA and incubated in 5% CO2 at 37°C. After 2 days, the supernatant was collected for cytokine analysis. After 7 days, [3H]-thymidine U0126 research buy was added and cells were harvested 10 h later. Cell proliferation was assessed by measuring [3H]-thymidine incorporation in a β-counter (Perkin Elmer, Waltham, MA, USA). Levels of IFN-γ, TNF and IL-6 in 2 days’ supernatants were assayed by cytometric bead array (CBA; BD Biosciences, San Jose, CA, USA) according to the manufacturer’s recommendations.
Samples were assayed using fluorescence activated cell sorter (FACS)Canto (BD Biosciences Pharmingen, San Jose, CA, USA) and analysed with FCAP Array Software (BD Biosciences). The limits of detection were 17·5 pg/ml for IFN-γ, 7·3 pg/ml for TNF and 5 pg/ml for IL-6. The effects of the three different diets were also evaluated in a Th2-driven airway hypersensitivity model (Fig. 1b) in a second set of animals. Mice were immunized on days 15 and 25 with intraperitoneal (i.p.) injections of 10 µg OVA mafosfamide absorbed onto 2 mg of Al(OH)3
(alum; Sigma). On day 33, the animals were anaesthetized briefly (Isofluran; Baxter Medical AB) and challenged with 100 µg of OVA in 25 µl of PBS by intranasal administration. This procedure was repeated on each of the following 4 days. Twenty-four hours after the final challenge, the mice were anaesthetized (xylazine 130 mg/kg and ketamine 670 mg/kg, i.p.). The chest was opened and blood was drawn by heart puncture. The blood sample was clotted and serum was collected after centrifugation (15 min at 3000 g). Bronchoalveolar lavage was performed by twice instilling 0·4 ml of PBS through the trachea followed by gentle aspiration. The proportion of eosinophils in the bronchoalveolar fluid was evaluated on slides prepared using a cytospin and stained with May–Grünwald/Giemsa. Sensitization was measured as OVA-specific IgE titres in the serum samples by passive cutaneous anaphylaxis . Mouse sera were diluted serially with PBS and 50 µl was injected intradermally into the shaved dorsal skin of anaesthetized (8 mg/kg xylazine and 40 mg/kg ketamine i.p.) Sprague–Dawley rats (Scanbur AB).