Ifn γ fitc 4s b3

Human cells were stimulated with 5 ng/ml PMA and 500 ng/ml ionomycin (Sigma-Aldrich) for 4 h at 37°C, with 2 μM monensin added for the final 3 h. Cells were surface stained on ice with the relevant markers and Zombie Aqua viability dye (BioLegend) for 30 min before fixing and permeabilizing using BD Cytofix/Cytoperm Kit as per the manufacturer’s instructions. The following Abs were used to stain intracellular molecules: IL-22/PECy7 (22URTI), IL-22/PE (22URTI), IL-4/PECy7 (8D4-8), IL-17A/APC (eBio64Dec17), IL-17F/aF488 (Poly5166), and IL-2/PerCP/Cy5.5 (MQ1-17H12), all from eBiosciences; IFN-γ/FITC (4S.B3) and IL-10/PE (JEs3-9D7) from BD Biosciences. Cells were washed a further two times in 2% FCS/PBS and then fluorescence was assessed using an NxT Attune or Fortessa.

Transduced T cells were co-cultured with target cells at a 1:2 ratio or with 3 μg/mL PMA (Sigma Aldrich) and 1 μg/mL Ionomycin (Sigma Aldrich) for 5 h in the presence of GolgiPlug Protein Transport Inhibitor (BD Biosciences). Cells were then stained for surface markers, fixed with Fixation Medium A (Thermo Fisher Scientific), washed, and stained with IL2-APC (MQ1-17H2, BD), TNFα-PE-Cy7 (MAB11, BD), IFNγ-FITC (4S.B3, BD), MIP1β-PerCP-Cy5.5 (D21-1351, BD), and IL-10-PE/Dazzle 594 (JES3-9D7, Biolegend) antibodies in Fixation Medium B (Thermo Fisher Scientific) for 20 min. Following a final wash, cells were analyzed on a BD Fortessa flow cytometer.