Glucose dose-response data were generated for each cell type and growth condition. Cells transfected with FLII12Pglu-700µΔ6 were treated with hexokinase inhibitor 3-bromopyruvic acid (BrPy, 100 µM) plus the respiratory chain complex I inhibitor rotenone (100 nM) for 30 min to inhibit glucose metabolism. During this time, cells were incubated with different glucose concentrations to equilibrate intracellular glucose with extracellular glucose before imaging as previously described to equilibrate intracellular and extracellular lactate for FRET measurement (33). FRET activity of FLII12Pglu-700µΔ6 was imaged as described above.