2.5 IFN-γ Elispot Assay

Elispot was performed using the human interferon-γ (IFN-γ)–precoated ELISPOT kit (DAKEWE, China) as suggested by the guidelines for users. The experimental step is similar to the previous paragraph. Key steps are as follows. We put the mixture of stimuli (50 μM peptide) and PBMC suspension (5 × 10⁵cells per well) into experimental wells. The plate was incubated in a 37°C 5% CO2 incubator for 12–48 h. Biotin-labeled antibody against IFN-γ was added to each well and incubated at 37°C for 1 h. Enzyme-labeled avidin (100 μl per well) was added and incubated at 37°C for 1 h. Finally, 100 μl per well of AEC substrate solution was added to the plate. Until spots emerge, we washed the plate three times with tap water and then count spots.