Apc conjugated anti cd8 mab

721.221 cells prepulsed with HIV-1 epitope peptide or 721.221 cells infected with HIV-1 virus were cocultured with T-cell clones or lines in a 96-well plate for 2 h at 37°C. Brefeldin A (10 μg/ml) was then added, and the cells were incubated further for 4 h at 37°C. The cells were then fixed with 4% paraformaldehyde and incubated in permeabilization buffer (0.1% saponin, 10% FBS, phosphate-buffered saline) and then were stained with APC-conjugated anti-CD8 MAb (Dako), followed by FITC-conjugated anti-IFN-γ MAb (BD Biosciences). The percentage of IFN-γ-producing cells among the CD8⁺ T-cell population was determined by using the FACS-Canto II.

HLA-B*52:01-RI8, -MI8, -SI8, or -WV8 peptide tetrameric complexes (tetramers) and HLA-A*24:02-RF10, HLA-C*12:02-IY11, -MY9, or -RL9 complexes were generated as previously described (58 (link)). CD8⁺ T cells specific for a given epitope were stained with phycoerythrin (PE)-conjugated epitope-specific tetramers at 37°C for 30 min. The cells were then washed twice with RPMI 1640 medium containing 5% FCS (R5), followed by staining with allophycocyanin (APC)-conjugated anti-CD8 MAb (Dako, Denmark) and 7-aminoactinomycin D (7-AAD; BD Pharmingen) at 4°C for 30 min. Finally, the cells were washed twice with R5 and then analyzed by using a FACS Canto II. Primed T cells unstained with tetramer were used as a negative control to determine gating of the tetramer⁺ population. The tetramer⁺ population was determined based on the gating of the negative control (<0% of tetramer⁺ cells).