Pe cy5 anti mouse cd3

Freshly isolated splenocytes or pulmonary immune cells were resuspended at a concentration of 1 × 10⁷cells/mL. Cells were stimulated with 10 μg/mL antigens (LppZ and Ag85A) or tuberculin PPD for 20 h or treated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 500 ng/mL Ionomycin (both from Sigma Aldrich) for 5 h. GolgiStop solution (BD Biosciences) was added to cell culture during the last 5 h of ex vivo stimulation. For surface staining, cells were incubated with PE-Cy5-anti-mouse CD3 (eBioscience), Pacific Blue-anti-mouse CD4 (Biolegend), APC-Cy7-anti-mouse CD8 antibodies, biotin-Annexin V and PerCP-streptavidin (BD Biosciences) for 30 min in the dark at 4°C. After washing with PBS containing 2% FBS, cells were fixed and permeated by the Cytofix/Cytoperm reagent from Fixation/Permeabilization kit (BD Biosciences). Cells were incubated with FITC-anti-mouse IFN-γ, PE-anti-mouse IL-2 (both from BD Biosciences), and APC-anti-mouse TNF-α (Biolegend) antibodies for 45 min at 4°C. Cells were washed, resuspended in PBS and acquired with a FACS CantoII flow cytometer (BD Bioscience) in 2 h. Data were analyzed by using FlowJo software 7.5 (Treestar Inc.).