Measuring Metabolic Activity in IDH1-Mutant Cells

HCT 116 cells, with and without pre-treatment of 1μM AGI-5198, were grown in 5% CO₂ at 37°C and OCR and ECAR was measured using an XFe96 analyzer (Seahorse Bioscience, North Billerica, MA, USA). HCT116 cells were plated in XF96 cell culture plates, 2.0*10⁴IDH1^WT/R132H HCT116 cells or 1.75*10⁴IDH1^WT/WT cells (to account for a more rapid proliferation of IDH1^WT/WT HCT116 cells relative to IDH1^WT/R132H HCT116 cells ([53 (link)] and our own observation), were seeded in each well of 96-well assay plates and incubated for 48 h prior to conducting the assay. For determination of OCR, medium was changed to DMEM supplemented with 10 mM glucose, 2 mM glutamine and 1.5 mM pyruvate. For the glycolytic flux, medium was changed to DMEM supplemented with 2 mM L-glutamine and the concentration of glucose added to initiate glycolysis was 25 mM. Four independent replicates were conducted with 10 technical replicates per cell line. Data were expressed as pmol of O₂ per minute and normalized by cell number measured by the CyQUANT Cell proliferation kit (Invitrogen™), which is based on a fluorochrome binding to nucleic acids. Fluorescence was measured in a microplate luminometer (ClarioStar BMG Labtech, Cary, NC, USA) with excitation wavelength at 485 ± 10 nm and emission detection wavelength at 530 ± 12.5 nm.