Ifn γ pe cy7 b27

Fresh post-REP TIL were washed in PBS and rested O/N without IL-2 in TIL-CM or if cryopreserved, thawed and rested O/N in the presence of 100 IU/mL of IL-2 followed by a pre-assessment incubation of 6 hours without IL-2. Rested TIL (0.5×10⁶) were incubated for 6 hours at 37°C with the CD107a (H4A3) flow cytometry antibody (BD Bioscience) in addition to phorbol myristate acetate (PMA)/ionomycin or TIL-CM alone (unstimulated control) in a 96-well plates. 1 hour into the incubation, the GolgiStop Monensin (BD Bioscience) was added. Post-incubation, cells were harvested and a surface staining was performed as described above using the CD3 FITC (SK7), CD4 PerCP-Cy5.5 (RPA-T4) and CD8 PB (RPA-T8). Cells were then fixed and permeabilized for intracellular staining using the BD Cytofix/Cytoperm (BD Bioscience). Cells were then blocked once again using goat serum and intracellularly stained with fluorochrome-conjugated antibodies against interferon (IFN)-γ PE-Cy7 (B27) and tumor necrosis factor (TNF)-α APC (Mab11) (BD Bioscience).
Samples were acquired using the BD FACS Canto II or BD LSRFortessa and analyzed using FlowJo Software (Tree Star). For surface stain analysis, gating was performed using fluorescence minus one when required. For functional assessment by flow cytometry, gating was performed by using the unstimulated condition.