Fitc conjugated anti ifn γ mab

PBMCs or purified NK cells were resuspended at 4 × 10⁶/mL with complete RPMI 1640 medium (consisting of 10% FBS, 100 U/mL penicillin and 100 U/mL streptomycin) and incubated with or without 2000 HA/mL inactivated A/PR8 virus in 96-well U-bottom plates for 18 hours. In some experiments, cells were also stimulated with different influenza virus subtypes, or cells were infected with RSV at a multiplicity of infection (MOI) of 1. For the last 4 hours, 1.77 μg/mL monensin (Sigma, St. Louis, MO) was added to all cultures. Cells were harvested, stained with surface markers as previously described, washed, fixed and permeabilized with 0.5% saponin. They were then stained with FITC-conjugated anti—IFN-γ mAb (BD Biosciences), washed and resuspended in PBS with 1% paraformaldehyde. Data were acquired using a flow cytometer.
In some experiments, cells were co-cultured with 10 μg/mL anti-NKp46 blocking antibodies (BD Biosciences) on ice for 1 hour. Cells were then stimulated for 18 hours with 2000 HA/mL A/PR8 or 3 μg/mL FluaRIX vaccine. A neutralizing rat anti—human IL-2 antibody (4 μg/mL) (BD Biosciences) was used to neutralize IL-2 in culture. The corresponding mouse IgG1, κ isotype and rat IgG2a, κ isotype antibodies were used as the respective controls for the anti-NKp46 and anti—IL-2 antibodies.

721.221 cells prepulsed with HIV-1 epitope peptide or 721.221 cells infected with HIV-1 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% fetal bovine serum [FBS]–phosphate-buffered saline [PBS]), after which they were stained with APC-conjugated anti-CD8 MAb (Dako, Denmark) 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 use of the FACSCanto II.

721.221 cells prepulsed with HIV-1 epitope peptide or infected with HIV-1 virus, or CD4⁺ T cells infected with HIV-1 virus were cocultured with HIV-1-specific bulk-cultured T cells or a T-cell clone in a 96-well plate for 2 h at 37°C. Brefeldin A (10 μg/mL) was then added, and the cells were incubated for another 4 h at 37°C. Next, the cells were fixed with 4% paraformaldehyde and incubated in permeabilization buffer (0.1% saponin, 10% FBS-phosphate-buffered saline [PBS]), after which they were stained with allophycocyanin (APC)-conjugated anti-CD8 monoclonal antibody (MAb; Dako, Denmark), followed by fluorescein isothiocyanate (FITC)-conjugated anti-IFN-γ MAb (BD Biosciences). The percentage of IFN-γ-producing cells among the CD8⁺ T-cell population was determined by FACS Canto II.