Fam flivo

For BrdU labeling, mice were injected I.P. with 1 mg BrdU daily for six days. One day later, tissues were harvested and BrdU incorporation in iNKT cells was evaluated with the BrdU Flow Kit (BD Pharmingen) per the manufacturer’s instructions. As a cytokine-driven model of iNKT cell activation, mice were injected I.V. with 2mg ultra-pure LPS from E. coli O111:B4 (InvivoGen) per kg body weight. As an antigen-driven model of iNKT cell activation, mice were injected I.P. with 1 μg αGalCer analog KRN7000 (Avanti Polar Lipids). Both αGalCer and LPS were prepared in DMSO and diluted 1:10 (v/v) in sterile saline immediately prior to injection. Mice were sacrificed and tissues harvested after either 4 or 72 hours. In some experiments, mice were injected I.P. with 200μg ultra-low endotoxin azide-free anti-IFNγ (XMG1.2) or isotype control antibodies (Biolegend) 8 hours prior to αGalCer injection. For in vivo analysis of apoptosis mice were injected I.V. with the fluorescent poly-caspase binding reagent FAM-FLIVO (Immunochemistry Technologies) and analyzed per the manufacturer’s instructions. For ex-vivo intracellular cytokine staining, suspensions of SVF cells were cultured for two hours at 37°C in complete media in the presence of Brefeldin A prior to staining with flow cytometry antibodies.

To measure mitochondrial membrane potential and mass, MitoTracker Deep Red FM and MitoTracker Green FM (Thermo Fisher) were loaded into single-cell suspensions for 20 min at 4 °C. To detect apoptotic cells in vivo during tumor challenge, the fluorogenic substrate FAM-FLIVO (ImmunoChemistry Technologies) was injected s.c. into tumor-bearing flanks of mice, and TDLNs were harvested for analysis 1 h later. To detect apoptotic cells in vitro during antigen processing and presentation, FAM-FLICA or SR-FLICA (ImmunoChemistry Technologies) was added to cells after 24 h culture and incubated for 1 h before analysis. Propidium iodide (0.05%, ImmunoChemistry Technologies) was also added to detect apoptosis. All extracellular flux assays were performed with Seahorse Bioscience XF96 Analyzers. Inhibitors were added at the following final concentrations: oligomycin (1 μM), FCCP (1.5 μM) and rotenone/antimycin A (0.5 μM).