Quantifying Antigen-Specific IFN-γ Secreting Cells

Enumeration of antigen-specific IFN-γ secreting cells was done by ELISpot assay, as described before [3 (link),5 (link),10 (link),25 (link)]. Briefly, spleen cells (4 × 10⁵/well) were added to ELISpot plates coated with an anti-IFN-γ antibody (Mabtech Inc., Cincinnati, OH, USA), followed by incubation in the presence of appropriate antigen-specific stimulant at a concentration of 2 µg/mL for 20 h at 37 °C, 5% CO₂. A peptide corresponding to CD⁸⁺ T cell epitope OVA_257-264: SIINFEKL (JPT Peptide Technologies GmbH, Berlin, Germany) was used as a stimulant. To measure background responses, cells were incubated in the absence of any stimulants. The plates were developed according to the manufacturer’s instructions. AEC substrate (Becton Dickenson, Franklin Lakes, NJ, USA) was added to visualize the spots. Spots were counted using an automated ELISpot plate reader (Cellular Technology LTD, Beachwood, OH, USA).