Golgi stop brefeldin a

For the identification of CD4⁺ Th cell subsets and CD8⁺ Tc cells, we used cytoplasmic cytokine staining method. Briefly, whole blood were diluted to 1:1 with saline solution and incubated with phorbol-12-myristate 13-acetate (PMA) (25 ng/ml), ionomycin (1 μg/ml) and Golgi Stop brefeldin A (10 μg/ml) (all from Sigma Aldrich, St. Louis, Missouri, USA) for 5 h at 37°C in 5% CO₂ milieu. The following monoclonal antibodies were used for cell surface staining: CD4-PE-Cy5 or CD8-PE-Cy5 (both from Beckman Coulter). The cells were then fixed and permeabilized with Intraprep™ permeabilization reagent (Beckman Coulter) according to the manufacturer’s instructions, and intracellular cytokines were stained with the combination of interferon (IFN)-γ-FITC, interleukin (IL)-4-PE, IL-10-PE (all from BD Biosciences) and IL-17-PE (R&D Systems, Minneapolis, MN, USA) monoclonal antibodies. Measurements were performed and data were analyzed on Coulter FC500 flow cytometer (Beckman Coulter) equipped with Kaluza 1.2a software. IgG1-FITC (BD Biosciences) and IgG1-PE (R&D Systems) isotype-matched antibodies were used during the identification. Cells were quantified as their percentage in the CD4⁺ or CD8⁺ lymphocyte population.

The distribution of Th cell subsets and Tc cells was assessed by flow cytometry. For stimulating cytokine-producing T cells, whole blood was diluted to 1:1 with saline solution and stimulated with phorbol-12-myristate 13-acetate (PMA) (25 ng/mL), ionomycin (1 μg/mL) for five hours at 37 °C in 5% CO₂ milieu. Golgi Stop brefeldin A (10 μg/mL) (all from Sigma Aldrich, St. Louis, MO, USA) was added to the culture for the last 4 h. After cell surface staining, cells were fixed and permeabilized with Intraprep^TM permeabilization reagent (Beckman Coulter) according to the manufacturer’s instructions. Then, intracellular cytokine staining was carried out with a combination of fluorophore-conjugated monoclonal antibodies. Cells were evaluated on a Coulter FC500 flow cytometer and data were analysed with Kaluza 1.2a software (both from Beckman Coulter). Isotype-matched antibodies were used in all experiments. All antibodies used for this measurement and the determined T cell subpopulations are summarized in Table 2.