Glycolysis stress test kit

A Glycolysis Stress Test kit and Seahorse XFe96 Bioanalyzer (Agilent, Santa Clara, CA) was used to measure metabolic flux in real-time. RTEC were seeded in a 24-well V7 PS cell culture microplate (Agilent Technologies; Santa Clara, CA) at a density of 3 × 10⁴ cells per 100 μL per well. Cells were monitored for adherence and left to grow overnight until the desired confluency was reached to obtain a consistent monolayer. Prior to the assay being performed, 1 mM l-glutamine was added to 100 mL of XF DMEM Medium, pH 7.4 (Agilent Technologies) and cells were then washed twice. The plate was placed in a non-CO₂ incubator at 37 °C for 1 h prior to the assay. Using the Glycolysis Stress Test kit (Agilent Technologies), each of the drug injection ports of the hydrated Sensor Cartridge was then loaded with 10 mM glucose, 1 μM oligomycin (simulant), 50 mM 2-deoxy-D-glucose (2-DG). Basal extracellular acidification rate (ECAR) was measured for 30 min followed by an assessment of glycolytic capacity. Where indicated, cells were treated with LPS (100 ng/ml) and/or metformin (1 mM) for 24 h.

The Extracellular Acidification Rate (ECAR) was measured using a Glycolysis Stress Test Kit and eXF96 Extracellular Flux Analyzer (Seahorse Bioscience) according to the manufacturer’s protocol. In brief, 10,000 cells were plated in 100 μl of their standard growing media and cultured overnight. On the day of the measurement, cells were washed with XF media and incubated in a CO2-free incubator at 37°C for 2 h to establish equilibration before loading. ECAR measurements were taken before and after the addition of glucose (10 mM), oligomycin (1 mM) and 2-DG (50 mM) and used to calculate glycolysis, glycolytic capacity and glycolytic reserve.

The Extracellular Flux Analyzer XF96 (Seahorse Bioscience, Billerica, MA, USA) was used to measure cellular glycolysis capacity using Glycolysis Stress Test Kit (Seahorse Bioscience), according to the manufacturer’s instructions. Cells (4 × 10⁴ cells/well) were seeded in XF96 cell culture microplates. For ECAR assays, 10 mM glucose, 1 μM oligomycin and 100 mM 2-deoxy-glucose (2-DG) were added to evaluate ECAR values. The values of ECAR was normalized based on the numbers of cells and the curve was drawn. For glucose uptake and lactate production, cells were separately cultured in 6-well plate and incubated for 24 h, subsequently, cell supernatant was collected to calculate the glucose concentration and lactate level by using Glucose Colorimetric assay kit (BioVision, USA) and Lactate Colorimetric Assay Kit II (BioVision, USA), respectively. The data were normalized to total cell number of each sample.