Absorbance was read at 405 nm on a microplate reader (Bio-Rad, Hercules, CA, USA). Mice were sacrificed 2 weeks after the last immunization and their spleens beta-catenin assay were removed. Spleen cells were released by mechanical dissociation, passing the tissue through stainless steel mesh and washing with Hank’s balanced salt solution (HBSS), N-2-hydroxyethylpiperazine 2-ethanesulfonic acid (Gibco BRL). Erythrocytes were lysed by incubation for 3 min in lysing solution (17 mm Tris–HCl, pH 7·65, 139·5 mm NH4Cl). Lysis was stopped by

adding HBSS-Hepes. Spleen cells were collected by centrifugation for 10 min at 800 × g and suspended in RPMI 1640 culture medium containing 2 mm l-glutamine (GIBCO), 100 units/100 μg/mL penicillin/streptomycin solution (GIBCO) and 10% heat inactivated foetal calf serum. Spleen cells (5 × 105 cells in 100 μL) from each group were plated in 96-well culture plates in RPMI 1640-Hepes culture medium, then 5 μL of each anti-mouse CD3+ UCHT1 IgG1 conjugated with R phycoerythrincyanin 5·1 (PC5), anti-mouse CD4+ (L3T4) H129·19 conjugated with R-phycoerythrin (R-PE) and anti-mouse CD8+ (Ly-2) 53-6·7 conjugated with fluorescence isothiocyanate (FITC) was added. PBS/BSA buffer was added to test

wells to bring the total volume in each well to 90 μL. After incubation at RT in dark for 15 min, 50 μL of PBS and 20 μL of foetal bovine serum were added sequentially to each JNK signaling pathway inhibitor well. Plates were centrifuged at 400 × g for 10 min at 4°C, supernatants were aspirated, and the cell pellets were resuspended in 200 μL of PBS/BSA for flow cytometric analysis with flow cytometry FC500 (Beckman coulter, Brea, CA, USA) using Cell Quest software (Becton Dickson, San Jose, CA, USA). Ten thousand events were collected per sample. The mouse immunization and the spleen cell isolation are the same as of described above.

Splenocytes were cultured in 96-well microtitre plates with soluble C. parvum extract or recombinant antigens 5 μg/mL in 0·2 mL of RPMI-1640 medium containing 5% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin, at 37°C in a 5% CO2 atmosphere for 3 days. Cell-free culture supernatants were harvested and were assessed for IFN-γ, IL-12 and IL-4 activities by standard ELISA as described previously (14). Briefly, the supernatant was serial diluted and coated to the 96-well microtitre plates at 100 μL/well. Then rat anti-mouse IFN-γ McAb (IgG1, BD Biosciences) or rat anti-mouse IL-12 (p70) McAb (IgG2b, BD Biosciences, San Jose, CA, USA) or IL-4 McAb (IgG1, BD Biosciences) diluted at 1 : 100 in PBS-4% BSA was added. After incubation for 1 h at 37°C, an alkaline phosphatase labelled rabbit anti-rat IgG conjugate (at 1 : 2000, Sigma) was used as detection reagent with the pNPP substrate (1 mg/mL, Sigma). Absorbance was read at 405 nm on a microplate reader (Bio-Rad).