2 deoxy d glucose

Real time bioenergetics analysis of extracellular acidification rates (ECAR) and oxygen consumption rates (OCR) of T-cells subjected to antibody stimulation was performed using the XF analyzer (Seahorse Biosciences). T-cells were cultured in serum free, unbuffered XF assay medium (Seahorse biosciences, Cat 102365-100) for 1 h. The cells were then seeded (6 × 10⁵/well) into the seahorse XF24 cell plates for analysis. Perturbation profiling of the use of metabolic pathways by T-cells was achieved by the addition of oligomycin (1 μM), FCCP (1 μM), Antimycin A (1 μM), rotenone (1 μM), D-glucose (10 mM), 2-deoxy-D-glucose (2DG, 50 mM; all from Seahorse biosciences, Cat# 103020-100 and 103015-100). Experiments with the Seahorse system were done with the following assay conditions: 2 min mixture; 2 min wait; and 4–5 min measurement. Metabolic parameters were calculated by Wave v2.4.1 Software. Experiments were done in at least triplicate wells.

To assess glycolytic function, we used an Agilent Seahorse XF Glycolysis Stress Test Kit (Seahorse Bioscience, #16814032, North Billerica, MA, USA) with a Seahorse XFe96 Analyzer to directly measure the real time extracellular acidification rate (ECAR). Before measurement, the sensor cartridge was hydrated in Seahorse XF Calibrant at 37 °C in a non-CO₂ incubator overnight. NGP, BE2 and NIH3T3 cells were seeded onto 6-well plates with different doses of DMAMCL, incubated for 24 h, and then the cells were collected, washed twice with cold PBS, and resuspended with phenol red-free assay solution. To avoid the impact of the different number of cells in each group on ECAR, we counted and seeded the same number of cells for each group onto the XF96 cell culture microplates (Seahorse Bioscience, North Billerica, MA, USA) precoated with Cell-Tak solution at the density of 4 × 10⁴ cells/well (NGP) and 3.5 × 10⁴ cells/well (BE2, NIH3T3). The cell culture microplates were centrifuged at 1000 rpm for 5 min to attach the cells and then glucose, oligomycin (ATP synthase inhibitors), and 2-deoxy-d-glucose (2-DG, hexokinase inhibitor) were added according to the manufacturer’s instructions (Seahorse Bioscience) and detected immediately.