Human ifn gamma elisa ready set go

IFNγ secretion of activated T cells was measured by a specific ELISA assay (Human IFN gamma ELISA Ready-SET-Go, eBioscience, 88-7316-88), after 6 h of activation with either M113 and M113-PD-L1^pos melanoma cell line or T2 cell line and its T2-PD-L1 transfected counterpart, loaded with 10µM of MELOE-1_36-44 peptide. The relative avidity of T cell clones and sorted T cells was measured by intracellular IFNγ and TNF-α production and CD107a membrane expression, in response to T2 cells loaded with a range of specific peptides (E/T ratio 1/2). After a 6-h-stimulation period with peptide-loaded T2 cells, in presence of brefeldin A at 10 μg/mL (Sigma, B7651), T cells were labeled with PE-conjugated specific anti-Vß antibodies (Beckman Coulter) and fixed with PBS 4% paraformaldehyde (VWR, 100504–858). Lymphocytes were then stained for cytokine production using APC conjugated anti-TNF-α (clone cA2, Miltenyi Biotec) and anti-IFNγ (clone 45–15, Miltenyi Biotec). Concerning CD107a labeling, specific T cells were stimulated at a E/T ratio of 1/2 with peptide loaded T2 cells for 4 h at 37°C in the presence of APC-conjugated mAb specific for CD107a (clone H4A3, BD Biosciences). T cells were then stained with selected anti-Vß antibodies (Beckman coulter) and analyzed by flow cytometry.