Fitc conjugated anti human cd8 mab

Isolated PBMCs or CD4⁺ T cells were resuspended at 1 × 10⁶/ml in RPMI-1640 medium containing 10% fetal bovine serum and stimulated with 50 ng/ml phorbol myristate acetate (PMA; Sigma-Aldrich) and 1 μg/ml ionomycin (Sigma-Aldrich) for 2 h. Subsequently, the cells were incubated for an additional 4 h in the presence of 1 μg/ml brefeldin-A (eBioscience, San Diego, USA) at 37°C in 5% CO₂. After incubation, the suspended cells were stained with relevant mAbs, including phycoerythrin-cyanin 5 (PE-Cy5)-conjugated anti-human CD3 mAb, fluorescein isothiocyanate (FITC)-conjugated anti-human CD8 mAb (eBioscience), PE-conjugated anti-human IFN-γ mAb, PE-conjugated anti-human IL-4 mAb (Miltenyi Biotec GmbH) and Alexa Fluor 488-conjugated anti-human c-MAF mAb (eBioscience). The data were analyzed using FlowJo 10 (Stanford University, San Francisco, USA). In this study, we defined CD3⁺ CD8⁻ IFN-γ⁺ cells as Th1 cells and CD3⁺ CD8⁻ IL-4⁺ cells as Th2 cells.

Fresh PBMCs or CD4⁺ T cells were resuspended in RPMI-1640 medium supplemented with 10% foetal bovine serum and stimulated with 50 ng/ml phorbol myristate acetate (PMA; Sigma–Aldrich) and 1 µg/ml ionomycin (Sigma–Aldrich) for 2 hours. Subsequently, the cells were incubated for an additional 4 hours in the presence of 1 µg/ml brefeldin-A (eBioscience, San Diego, USA) at 37°C with 5% CO₂. The suspended cells were subsequently stained with relevant mAbs, including phycoerythrin-cyanin 5 (PE-Cy5)-conjugated anti-human CD3 mAb, fluorescein isothiocyanate (FITC)-conjugated anti-human CD8 mAb (eBioscience), PE-conjugated anti-human IL-17A mAb, and PE-conjugated anti-human IL-23R mAb (R&D Systems, Minnesota, USA). FlowJo 10 (Stanford University, San Francisco, USA) was used to analyse the data. In this study, we defined CD3⁺ CD8^- IL-17A⁺ cells as Th17 cells.