IFN-γ ELISPOT assay

The frequency of IFN-γ-producing splenocytes was analyzed with the ELISPOT assay (Mabtech, Stockholm, Sweden). Briefly, a total number of 4 × 10⁵ spleen cells were seeded on each well of 96-well and in vitro stimulation was carried out with 10 μg/ml of P24-Nef peptide, as positive control cells stimulated with PHA and wells containing un-stimulated cells and RPMI 1640 were used as negative controls. The plates were incubated at 37 °C in 5% CO₂ for 24 h. After in vitro re-stimulation, the plates were washed five times with washing buffer and 100 μl of anti-mouse IFN-γ in PBS containing 0.5% FBS was added to the wells and incubated for 2 h at room temperature. The plates were washed five times with washing buffer and incubated for 1hr at room temperature with 100 μl of 1/1000 diluted streptavidine-conjugated alkaline phosphatase. After final wash with washing buffer, spots were developed by adding 100 μl of BCIP/NBT substrate to wells and incubating 30 min at room temperature in the dark. The plates were then rinsed three times with distilled water and dried at 4 °C. The spots were counted by stereo microscope (Nikon, Japan). The number of specific IFN-γ-producing lymphocytes was calculated by subtracting the spots from stimulated wells with un-stimulated one.