Ifn γ clone 4s b3

UV1-stimulated T cells were thawed and resuspended in X-vivo 15 medium (Lonza) before co-culture with either autologous Epstein Barr virus transformed B-cell lines (EBV-LCLs) or autologous PBMCs loaded with CellTrace Violet (Life Technologies Carlsbad, CA, USA) for gating purposes and loaded or not with the UV1 mix of peptides (10µM). E:T ratio was 1:2. Cells were co-cultured for 10 h in the presence of GolgiStop and GolgiPlug (both BD Biosciences, San Jose, CA, USA) at a dilution of 1/1000. After 10 h, cells were harvested, washed in staining buffer consisting of phosphate buffered saline (PBS) containing 2% Fetal Calf serum (FCS) before staining using the PerFix-nc kit (Beckman Coulter, Brea CA, USA) following the manufacturer’s instructions. The following antibodies were used: anti-CD4-PECy7 (clone RPA-T4), CD8-BV-605 (clone RPA-T8, BioLegend, San Diego CA, USA), CD3-APC (clone OKT3) TNF-α-PE (clone MAb11, BD Biosciences), and IFN-γ (clone 4S.B3) All antibodies were purchased from eBioscience, except where otherwise noted. Cells were acquired on a FacsCanto flow cytometer and the data were analyzed using FlowJo software (Treestar Inc. Ashland, OR, USA). Cells were gated on lymphocytes in forward scatter (FSC) vs. side scatter (SSC), single cells, CellTrace negative cells, then CD4+CD3+ cells or CD8+CD3+ cells.