Quantifying Antigen-Specific IFN-γ Response

The frequencies of antigen-specific IFN-γ-secreting cells were evaluated using a mouse ELISPOT kit (BD Biosciences), as previously described [12 (link),23 (link)]. Briefly, 14 days after the last vaccination, splenocytes were obtained from the immunized mice and then plated at 5 × 10⁵ cells/well onto purified anti-IFN-γ-coated ELISPOT plates. The cells were treated with 0.5 µg/well of OVA_257–264 peptides (Anaspec, San Jose, CA, USA) or the 500 median tissue culture infectious dose (TCID₅₀)/well of UV-inactivated pH1N1 influenza virus for 3 days at 37 °C. The spot-forming units (SFUs) of antigen-specific IFN-γ-secreting cells were calculated using an ELISPOT plate reader (Cellular Technology Ltd., Cleveland, OH, USA).