mMCPT-1 was quantified in serum collected 1 hour after the second challenge according to the manufacturer’s protocol (eBioscience). BLG-specific ELISAs were performed using protocols modified from ref. 7 (link). Briefly, plates were coated overnight at 4°C with 100μg/mL BLG in 100mM carbonate-bicarbonate buffer (pH 9.6). Plates were blocked for 2 hours at room temperature with 3% BSA. Samples were added in 1% BSA and incubated overnight at 4°C. Assays were standardized with BLG-specific antibodies (IgE or IgG1) purified on a CNBr-Sepharose affinity column from mice immunized with BLG+alum 42 (link). BLG-specific antibodies were detected with goat anti-mouse IgE-UNLB (Southern Biotech) and rabbit anti-goat IgG-AP (ThermoFisher) then developed with p-NPP (KPL Labs) or IgG1-HRP (Southern Biotech) and developed with TMB (Sigma).
For cytokine analysis spleens were harvested 24h post challenge from A. caccae or CMA colonized mice sensitized with BLG+CT for 5 weeks. Splenocytes were stimulated at a concentration of 2×106 cells/ml at 37°C, 10% CO2 with 10 mg/ml BLG (Sigma) in cDMEM (with 4% FCS (HyClone), 10mM HEPES (Gibco), 100U/ml Penicillin/Streptomycin (Gibco) and 55μM 2-mercaptoethanol (Gibco). Cytokine concentrations in 72 hr culture supernatants were determined by ELISA for IL-13 and IL-4 (both from Invitrogen).
For cytokine analysis spleens were harvested 24h post challenge from A. caccae or CMA colonized mice sensitized with BLG+CT for 5 weeks. Splenocytes were stimulated at a concentration of 2×106 cells/ml at 37°C, 10% CO2 with 10 mg/ml BLG (Sigma) in cDMEM (with 4% FCS (HyClone), 10mM HEPES (Gibco), 100U/ml Penicillin/Streptomycin (Gibco) and 55μM 2-mercaptoethanol (Gibco). Cytokine concentrations in 72 hr culture supernatants were determined by ELISA for IL-13 and IL-4 (both from Invitrogen).