2.6. ELISpot Assay

Enumeration of antigen-specific IFN-γ secreting cells was performed by ELISpot assay as previously described [15,17,21]. Briefly, spleen cells (at a final cell density of 4 × 10⁵/well) were added to ELISPOT plates coated with an anti-IFN-γ antibody (Mabtech Inc., Cincinnati, OH, USA), and incubated in the presence of appropriate antigen-specific stimulant at a concentration of 2 µg/mL for 20 h at 37 °C, 5% CO₂. For OVA protein-immunized animals, CD8 T cell epitope OVA_257–264: SIINFEKL or CD4 T cell epitope OVA_323–339: ISQAVHAAHAEINEAGR peptides (JPT Peptide Technologies GmbH, Berlin, Germany) were used as stimulants. Cells were also incubated without any stimulants to measure background responses. The plates were then incubated, washed and developed according to the manufacturer’s instructions. AEC substrate (Becton Dickenson, Franklin Lakes, NJ, USA) was used to visualize the spots. Spots were counted using an automated ELISpot plate reader (BIOSYS, Miami, FL, USA).