Sera were collected on day 0 prior to immunization and days 3, 7, 14 after immunization. Mice were also immunized i.p. or s.c. with 100 μg TNP-OVA (Biosearch Technologies) absorbed in 4 mg alum (Sigma-Aldrich) on days 0 and 21. Sera were collected on day 0 prior to immunization and see more days 7, 14, 21, 28, and 35 after immunization. Total immunoglobulin levels were determined by ELISA, as

described previously 43. Briefly, total IgM, IgG3, IgG2c, IgG1, and IgE were captured by plate-bound goat anti-mouse IgM, IgG, or IgE and detected with alkaline phosphatase-conjugated goat anti-mouse IgM, IgG3, IgG2c, IgG1, and IgE (Southern Biotechnology Associates), respectively. A standard curve was prepared using known quantities of BH8 (anti-PC IgM, generated in our laboratory) or anti-TNP Ab (IgG1, eBioscience). To measure specific anti-PC or anti-TNP Abs concentration, plates were coated with PC-BSA or TNP-BSA. p-Nitrophenyl phosphate (Sigma-Aldrich) was added, and color development was determined on a Titertek Multiskan Plus reader (Labsystems, selleck chemicals llc ICN Biomedicals) at 405 nm. The 96-well high-binding plates

were coated with goat anti-mouse IgG or TNP-OVA and single-cell splenic suspensions were prepared 7 days after primary or secondary TNP-OVA/Alum immunization. In addition, 1×106 total splenocytes were seeded in each well containing 100 μL cRPMI followed by a 1:3 serial dilution. Cells were incubated at 37°C for 24 h before being lysed with PBS containing 0.05% Tween 20. Alkaline phosphatase-conjugated goat anti-mouse IgG1 was added and spots visualized by 5-bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich) and counted under a dissection microscope. Spots were then dissolved in 50 μL DMSO and absorbance of each well was measure with a spectrophotometer at 650 nm. RT-PCR was performed as described previously 41. Briefly, total RNA was isolated using TRIzol (Invitrogen), cDNA was generated using the Omniscript RT-PCR kit (Qiagen), and PCR was performed using GoGreen Taq master mix (Promega)

or SYBER green GPX6 master mix (Invitrogen) at an annealing temperature of 60°C for 30–35 cycles. The following primer pairs were used: β-actin: 5′-TACAGCTTCACCACCACAGC-3′ and 5′-AAGGAAGGCTGGAAAAGAGC-3′; Camp: 5′-CGAGCTGTGGATGACTTCAA-3′ and 5′-CAGGCTCGTTACAGCTGATG-3′; CD19: 5′- GGAGGCAATGTTGTGCTGC-3′ and 5′- ACAATCACTAGCAAGATGCCC-3′; CD3e: 5′-ATGCGGTGGAACACTTTCTGG-3′ and 5′-GCACGTCAACTCTACACTGGT-3′; IL-4: 5′-ACCACAGAGAGTGAGCTCG-3′ and 5′-ATGGTGGCTCAGTACTACG-3′. Purified splenic naïve CD4+ T cells (0.5×106 cells/mL) were obtained using negative selection followed by a CD62L+ magnetic bead selection (Miltenyi Biotec) and stimulated with 2 μg/mL plate-bound anti-CD3 and 2 μg/mL anti-CD28 (eBioscience). Cells were cultured in 96-well flat-bottom plates in 200μL of cRPMI with 1 ng/mL recombinant mouse IL-4, 10 ng/mL recombinant mouse IFN-γ, 5 μg/mL anti-IL-12 antibody (eBioscience), in the presence or absence of 100–1000 ng/mL mCRAMP peptide.