Reactive oxygen species (ROS), mitochondrial membrane potential (MMP), mitochondrial mass, and Annexin
For analysis of intracellular ROS, TCR-T cells were incubated with CellROX Orange at 5 µM in complete culture media for 30 minutes at 37°C. For detection of effector TCR-T cells intracellular ROS level, TCR-T cells were incubated with MAGE-B2 peptide-loaded T2 Luc Red cells at E:T ratio=1 for 24 hours, cell mixtures were then incubated with CellROS Orange at 5 uM in complete culture media for 30 minutes at 37°C. Cells were then labeled with human antibody against CD3 and CD8 for 15 minutes at 4°C, and ROS level was determined by flow cytometry analysis using mean fluorescence intensity of CellROS Orange gated on CD8+ and CD3+ double positive cells.
Similarly, MMP and mitochondrial mass was measured by labeling cells either by themselves or after co-cultured with T2 Luc Red cells at E:T ratio=1 for 24 hours with DilC1(5) at 50 nM for 15 minutes at 37°C or MitoTracker Red CMXRos at 25 nM for 15 minutes at 37°C, respectively. Cells were then labeled with antibodies specific for CD3+ and CD8+, followed by flow cytometry analysis.
For analysis of Annexin V, TCR-T cells or cell mixtures of TCR-T cell and MAGE-B2 peptide-loaded T2 Luc Red cells after 24-hour co-incubation were labeled with antibodies specific for CD4+ and CD8+ then washed and labeled with FITC Annexin V in Annexin V Binding Buffer at 1e6 cells/ml for 15 minutes at room temperature in the dark. Cells were diluted with Annexin V Binding Buffer to 0.2e6 cells/mL and analyzed by flow cytometry.