PBMCs from HIV-1-infected individuals were expanded by culture with each HIV-1 peptide for 2 weeks. .221-C1202 cells prepulsed with each HIV-1 peptide or .221-C1202 cells infected with HIV-1 NL4-3 were added to a 96-well plate together with bulk-cultured T cells, and the cells were incubated for 4 h at 37°C with brefeldin A (10 μg/ml). The cells were then stained with peridinin chlorophyll protein (PerCP)-labeled anti-CD3 mAb (BioLegend), phycoerythrin (PE)-labeled anti-CD4 mAb (BioLegend), and allophycocyanin (APC)-labeled anti-CD8 mAb (Dako) and subsequently fixed with 4% paraformaldehyde and incubated in permeabilization buffer (0.1% saponin–10% FBS–PBS). Thereafter, the cells were stained with FITC-labeled anti-IFN-γ mAb (BioLegend). Staining data were acquired on a FACSCanto II instrument (BD Biosciences) and analyzed using FlowJo 10.5.3 software.
Fitc labeled anti ifn γ mab
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FITC-labeled anti-IFN-γ mAb is a monoclonal antibody conjugated with the fluorescent dye FITC (Fluorescein Isothiocyanate). It binds to and detects interferon-gamma (IFN-γ), a cytokine produced by various immune cells.
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2 protocols using fitc labeled anti ifn γ mab
1
Quantifying HIV-1-specific T Cell Responses
2
Induction of LY6K-reactive CTLs from HLA-A24+ Donor
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Induction of LY6K177–186-A24 SP-reactive CTLs from an HLA-A24+ and HLA-DR15+ human donor (HD3) was performed by stimulating donor-derived lymphocytes with LY6K177–186-A24 SP, as previously described.24 (link),46 (link) Monocyte-derived DCs were generated from CD14+ cells by in vitro culture, as described previously.23 (link) Autologous immature DCs were maintained alive or fixed for 3 min in 0.1% glutaraldehyde (Sigma-Aldrich) for use as controls. DCs were pulsed with 16 μM each LY6K177–186-A24 SP, LY6K172–191-LP, or control LP for 3 h, and washed 3 times. 0.1 KE/mL OK432 was added during and after the peptide pulse to induce the maturation of DCs. LY6K119–142-LP, which does not include a known CTL-epitope, was used as a control LP. LY6K177–186-A24 SP-reactive bulk CTLs were added at a 2:1 ratio for 6 h in medium containing 10 μg/mL brefeldin A (Sigma-Aldrich). IFNγ production by LY6K177–186-A24 SP-specific CTLs was measured by intracellular labeling. The cells were stained with a fluorescein isothiocyanate (FITC)-labeled anti-IFNγ mAb (BioLegend) in combination with a PerCP-labeled anti-CD8 mAb (BioLegend) and a PE-labeled HLA-A24/LY6K177–186-specific tetramer.
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