Anti ifn γ apc efluor 780

For intracellular cytokine staining of YF-tetramer⁺ CD8⁺T-cells, PBMCs of tetramer reactive samples were stimulated for 6 hours with phorbol myristate acetate (PMA) and ionomycin. One to two million cells were incubated in medium consisting of RPMI-1640 with 10% fetal calf serum (FCS) in the presence of PMA (10 ng/mL) and ionomycin (1 ng/mL), anti-CD107a FITC (eBioscience), brefeldin A (10 microg/mL; Invitrogen), GolgiStop (BD Biosciences) and co-stimulation (anti-CD28) for 6 hours at 37°C and 5% CO2. As a control, the same conditions without PMA and ionomycin were used. After incubation, 20 μL of tetramer mix was added to the samples in a 96-wells plate for 30 minutes at 4°C. Subsequently, 30 μL of antibody mix with CD3 V500 (Invitrogen) and CD8 BV785 from Biolegend were added for 30 minutes. The Cytofix/Cytoperm reagent (BD Biosciences) was used for fixation and permeabilization. After permeabilization, the following monoclonal antibodies were added: anti-TNF-α AF700, anti-IL-2 PE (BD Biosciences), anti-Mip1-β PE-Cy7 (Biolegend), anti-IFN-γ APC-eFluor 780 (eBiosciences). Cells were analyzed by LSR Fortessa and FlowJo v. 9.7.6 software (Stanford University, 1995–1996).