2 n 7 nitrobenz 2 oxa 1 3 diaxol 4 yl amino 2 deoxyglucose 2 nbdg

To measure metabolic parameters, activated NKT cells (1 × 10⁵) were incubated with different reagents as indicated in the figure legends. To measure mitochondrial mass and potential, cells were incubated with 30 nM MitoTracker™ Green (Invitrogen) and 60 nM of tetramethylrhodamine methyl ester perchlorate (TMRM) (Invitrogen), respectively, for 30 min at 37°C in RPMI 1640 complete media. Mitochondrial ROS levels were measured by incubating cells in 2.5 μM MitoSOX (Invitrogen) for 30 min at 37°C in RPMI 1640 complete media. To measure total cellular ROS, activated NKT cells were incubated with 1 mM 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) (Invitrogen) in RPMI complete media for 30 min at 37°C. To measure glucose uptake, cells were incubated in 2-(N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl) amino)-2-deoxyglucose (2-NBDG) (Invitrogen) (20 μM) for 1 h at 37°C in glucose-free RPMI 1640 media containing 10% dialyzed FBS. To measure GSH, cells were stained using an intracellular glutathione detection assay kit (Abcam) for 20 min at 37°C in RPMI 1640 complete media. Cells were stained for surface antigens and acquired on a FACS Canto II (BD Biosciences).