Capture anti murine ifn γ or il 4 antibodies

Capture anti-murine IFN-γ or IL-4 antibodies (R&D Systems, Minneapolis, MN, USA) were individually coated by overnight incubation at 4 °C onto High-Protein Binding IP 96-well Multiscreen TM plates (Millipore, Bedford, MA, USA). Plates were blocked with 1% BSA for 2 h. Then, 2 × 10⁵ splenocytes per well were seeded in RPMI, and stimulated overnight (for IFN-γ) and 48 h (for IL-4) at 37 °C, in 5% CO₂ with the VVWR L3 peptide-mix (JPT Peptide Technologies, Berlin, Germany). Concanavalin A (Con A, 5 mg/mL; Sigma-Aldrich, St. Louis MO, USA) and RPMI were used as a positive and negative controls, respectively. After stimulation, plates were washed and incubated overnight at 4 °C with biotinylated anti-mouse IFN-γ or IL-4 antibodies. Spot development was assessed after the additon of 5-Bromo-4-Chloro-3’ Indolylphosphate p-Toluidine Salt (BCIP) and Nitro Blue Tetrazololium Chloride (NBT) (R&D Systems, Minneapolis, MN). An automated ELISPOT reader system (CTL analyzers, Cleveland OH, USA) was used for spot quantification using the ImmunoSpot software (CTL analyzers, Cleveland OH, USA). The mean number of spots from triplicate wells was adjusted to 1×10⁶ splenocytes. Antigenspecific responses to IFN-γ and IL-4 were obtained after deducting the number of spots formed in the wells containing the RPMI alone from the spots developed in response to the L3 peptides.