Anti cd8 apc cy7

Purified CD8⁺ T cells with or without IL-35 stimulation were incubated in the presence of anti-CD8 APC Cy7 (eBioscience) and anti- programmed death-1 (PD-1) FTIC (eBioscience) for surface staining, and anti- cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) PE (eBioscience) for intracellular staining. In certain experiments, purified CD8⁺ T cells were stimulated with either PMA (50 ng/mL)+ionomycin (1 μg/mL) or AFP peptide in the presence of monensin (10 μg/mL) for 6 h. Cells were transferred to FACS tubes, and anti-CD8 APC Cy7 (eBioscience) was added for a 20 min incubation at 4°C in the dark. Cells were then stained with anti-IFN-γ APC (eBioscience) for 20 min at room temperature after fixation and permeabilization. Isotype controls were used to enable correct compensation and confirm antibody specificity. Acquisitions were performed using Cell Quest Pro Software (BD Biosciences Immunocytometry Systems, San Jose, CA, USA) in a FACS Calibur analyser (BD Biosciences Immunocytometry Systems). Data were analyzed using FlowJo Software Version 8.4.2 for Windows (Tree Star, Ashland, OR, USA).

Three weeks after the final immunization of antigens, single-cell suspensions (1 × 10⁶/mL) from the lung and spleen of adjuvant-, BCG-, and antigen-immunized mice were stimulated with purified proteins (InsB, 2 μg/mL; ESAT-6, 2 μg/mL) for 24 h at 37°C. IFN-γ cytokine levels were analyzed in the culture supernatant via sandwich enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol. Additionally, single-cell suspensions were stimulated with purified proteins (InsB; 2 μg/mL, ESAT-6; 2 μg/mL) for 12 h at 37°C in the presence of GolgiStop (eBioscience, San Diego, CA, United States), and then the cells were stained with Live/Dead Stain (InvivoGen, San Diego, CA, United States) and with anti-CD4 (PerCp-Cy5.5, eBioscience), anti-CD8 (APC-Cy7, eBioscience), and anti-CD3 (BV421, eBioscience) antibodies for 30 min at 4°C. Next, the stained cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA, United States) according to the manufacturer’s protocol and then stained with anti-IFN-γ (PE, eBioscience). The cells were analyzed with a FACSVerse flow cytometer using FlowJo software.