Pe cy7 conjugated tnf α mabs

RF10-specific CD8⁺ T cell lines were stimulated with C1R-A*2402 cells prepulsed with 100 nM RF10 peptide for 2 h at 37 °C. Brefeldin A (10 μg/ml, Sigma-aldrich) was added, and the cells were then incubated further for 4 h at 37 °C. Subsequently, they were stained with 7-AAD and Pacific blue-conjugated anti-CD8 mAb, fixed with 4% paraformaldehyde solution at 4 °C for 20 min, and then made permeable with 0.1% saponin buffer at 4 °C for 10 min. Thereafter, the cells were stained with FITC-conjugated anti-IFN-γ mAb (BD Pharmingen), APC-conjugated IL-2 mAbs (BD Pharmingen), PE-Cy7-conjugated TNF-α mAbs (BD Pharmingen), and APC-H7-conjugated MIP-1β mAbs (BD Pharmingen). Nonspecific production of cytokines was excluded by subtracting the data of the negative control, which was the same sample stimulated with C1R-A*2402 cells without the peptide and stained with the same mAbs. Poly-functionality was quantified as a standard index according to the formula: polyfunctionality index = F₀ × 0/4 + F₁ × 1/4 + F₂ × 2/4 + F₃ × 3/4 + F₄ × 4/4, where F_i is the frequency of cells expressing i functions (i = 0, 1, 2, 3, or 4) [41 ].