2 deoxyglucose 2 nbdg

LβT2 cells were grown in complete media (4.5 g/l glucose with sodium pyruvate) supplemented with antibiotics and 10% fetal bovine serum. Cells were harvested after a PBS wash with 0.25% trypsin, washed with PBS again and stained in PBS + Zombie NIR (Biolegend, Cat # 423106) diluted 1:1,600 for 20 min on ice protected from light. Cells were pelleted and resuspended in serum free DMEM (4.5 g/l glucose with sodium pyruvate), 1 × 10⁶ cells/200 μl/FACS tube. 10 nM GnRH was added to cells 30 min before flow cytometry analysis. Immediately prior to acquisition, 300 μM 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) (ThermoFisher Scientific) (2-NBDG) (Life Technologies, cat # N13195) was added to samples. Samples were acquired on slow for 1 min, followed by medium for 30 s, then high for 30 s to monitor that rate of increase in 2-NBDG fluorescence and ensure that the signal became saturated. The rate of glucose uptake was determined by the slope of the line produced by 2-NBDG mean fluorescence intensity plotted vs time (time gate = 15 s to 45 s). Samples were acquired in technical triplicates on a FACSCanto II flow cytometer (BD Biosciences, NJ, USA) and analyzed with FlowJo Software v 10.6.2 (Treestar, Ashland, OR, https://www.flowjo.com/).

(2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) (100 µM; ThermoFisher Scientific) with Hoechst (1 µg mL⁻¹) in low-glucose media (5 mM glucose, 10% FBS) was diffused into the chamber for 30 min at 37 °C at the desired time after chamber top placement (e.g., 2 h or 7 days). This dye was often combined with tetramethylrhodamine, ethyl ester (TMRE) (1 nM; ThermoFisher Scientific) in order to obtain simultaneous live-cell metabolic measurements. Chambers were removed, rinsed three times in 1× PBS, and immediately imaged using standard excitation and emission filters at ×20 on the Nikon Eclipse Ti widefield fluorescence microscope or on a Zeiss 710 Laser Scanning Confocal Microscope with a ×10 dry objective.