Seahorse xf glycolysis stress test kit

Cellular oxygen consumption and extracellular acidification were measured with a Seahorse XF extracellular flux analyzer according to the manufacturer’s instructions (Agilent). PASMCs were cultured in a Seahorse XF 24-well assay plate in antibiotic-free medium at a density of 20,000 cells/well, and were treated with siRNA for p53 (si-TP53; Invitrogen) and negative control siRNA (Invitrogen) after attachment. The medium was replaced with antibiotic-containing medium after 24 hours. After incubation for a further 24 hours, the plates were washed and the medium was replaced with pre-warmed running medium (XF base medium supplemented with 10% D-glucose, 100 mM pyruvate, and 200 mM glutamine for the mitochondrial stress test, or with 200 mM glutamine alone for the glycolysis stress test). Then incubation was performed in a non-CO₂ incubator for 60 min at 37°C. The basal oxygen consumption rate and extracellular acidification rate were recorded for 24 min, followed by performance of the mitochondrial stress test (1μM oligomycin, 2 μM FCCP, and 0.5 μM rotenone/antimycin A) and glycolysis stress test (10 mM glucose, 1 μM oligomycin, and 50 mM 2-DG). All reagents were obtained from the Seahorse XF Cell Mito Stress Test Kit (Seahorse Bioscience, #103015–100) and the Seahorse XF Glycolysis Stress Test Kit (Seahorse Bioscience, #103020–100).

The Seahorse XF Glycolysis Stress Test Kit and the Seahorse XF^e24 Extracellular Flux Analyzer (Seahorse Biosciences) were used to determine the glycolytic function in cells. The Seahorse XF Cell Mito Stress Test Kit (Seahorse Biosciences) was used to determine the cellular mitochondrial capacity. Cells seeded into a 24‐well plate (1 × 10⁴ cells/well) for overnight incubation were treated with fresh medium containing various concentrations of β‐elemene for 24 hours before the test operation. For Seahorse assay, the cells were washed with Seahorse assay medium. Next, interval injections of 10 μmol/L oligomycin, 2.5 μmol/L FCCP and 5 μmol/L rotenone/antimycin A were for determining the oxygen consumption rates (OCR). Then, 100 mmol/L glucose, 10 μmol/L oligomycin and 500 mmol/L 2‐deoxy‐glucose (2‐DG) were in turn added to determine the extracellular acidification rates (ECAR). The value of OCR and ECAR was calculated by normalization to the cell number.

The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in cells were examined using a Seahorse XF Cell Mito Stress Test Kit and a Seahorse XF Glycolysis Stress Test Kit, respectively, on a Seahorse XF-96 metabolic flux analyzer (Seahorse Bioscience, North Billerica, MA, USA). In short, OS cells were loaded onto the XF-96 cell culture microplates at 3 × 10⁴ cells per well. After probe calibration, for OCR, each well was added with 1 mM oligomycin, 1 mM FCCP, 2 mM antimycin A and 2 mM Rotenone (A&R); for ECAR, each well was added with 10 mM glucose, 1 mM oligomycin and 80 mM 2-DG. The data were evaluated and analyzed using the Seahorse XF-96 Wave software.